Multivalent carriers of bi-specific antibodies

ABSTRACT

Provided herein are targetable constructs that are multivalent carriers of bi-specific antibodies, i.e., each molecule of a targetable construct can serve as a carrier of two or more bi-specific antibodies. Also provided are targetable complexes formed by the association of a targetable construct with two or more bi-specific antibodies. The targetable constructs and targetable complexes of the invention are incorporated into biosensors, kits and pharmaceutical compositions, and are used in a variety of therapeutic and other methods.

This application claims priority to provisional application Ser. No.60/483,832, filed Jul. 1, 2003, the contents of which are incorporatedherein in their entirety.

FIELD OF THE INVENTION

The invention relates to compounds that are multivalent carriers ofbi-specific antibodies (bsAbs), i.e., each molecule of the compound canserve as a carrier of two or more bi-specific antibodies. The inventionfurther relates to complexes formed by the association of a multivalentcompound with two or more bi-specific antibodies. In preferredembodiments, the compounds of the invention form complexes that havedesirable attributes such as increased affinity, high stability in vitroand/or in vivo, and preferred pharmacokinetics. The compounds andcomplexes of the invention are useful for therapy and in vitroapplications.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

An approach to cancer therapy and diagnosis involves directingantibodies (Abs) or antibody fragments to disease tissues, wherein theantibody or antibody fragment can target a therapeutic agent to thedisease site, i.e., a targeted tissue. A “targeted tissue” is anybiological entity (e.g., a system, organ, tissue, cell, organelle,receptor, surface antigen, transmembrane protein, secreted polypeptide,or intracellular component) to which a targetable construct ispreferentially delivered. The term “delivered” encompasses beingcontacted with, bound to, and/or internalized by, a targeted tissue. Asused herein, the term “and/or” has the meaning of “and, additionally oralternatively” or “and, in addition to or in the alternative.”

In therapeutic aspects of the invention, the targeted tissue ismalignant, infected, inflamed (as in certain autoimmune diseased sites),dysfunctional or displaced or ectopic (e.g., infected cells, cancercells, endometriosis, etc.), or otherwise diseased (e.g.,atherosclerosis, ischemia, clots). Antibodies are used to delivertherapeutic agents to the targeted tissue.

The use of a bsAb/low molecular weight (MW) hapten system is not withoutsome limitations. The arm of the bsAb that binds to the low MW haptenmust bind with high affinity, since a low MW hapten is desirably onethat clears the living system rapidly when not bound by bsAb. Thenon-bsAb-bound low MW hapten needs to clear the living system rapidly toavoid non-target tissue uptake and retention. Moreover, the therapeuticagent must remain associated with the low MW hapten throughout itsapplication within the bsAb protocol employed.

This application incorporates by reference the entirety of U.S.application Ser. No. 09/337,756 (Docket No. IMM 138; Atty Docket No.018733-0884), entitled “Use of bi-specific antibodies for pre-targetingdiagnosis and therapy”, which was filed Jun. 22, 1999.

This application incorporates by reference the entirety of U.S.application Ser. No. 09/382,186 (Docket No. IMM 138 CIP; Atty Docket No.018733-0942), entitled “Use of bi-specific antibodies for pre-targetingdiagnosis and therapy”, which was filed Aug. 23, 1999.

This application incorporates by reference the entirety of U.S.application Ser. No. 09/823,746 (Docket No. IMM 160; Atty Docket No.018733-1015), entitled “Production and use of novel peptide-based agentsfor use with bi-specific antibodies”, which was filed Apr. 3, 2001.

This application incorporates by reference the entirety of U.S.Provisional Application Ser. No. 60/361,037 (Docket No. IMM 169; AttyDocket No. 018733-1037), entitled “Bispecific antibody point mutationsfor enhancing rate of clearance”, which was filed Mar. 1, 2002.

This application incorporates by reference the entirety of published PCTApplication WO 99/66951 by Hansen et al., entitled “Use of bi-specificantibodies for pre-targeting diagnosis and therapy”, which describessome of the reagents used in the Examples herein.

The synthesis of IMP 192 is described in Karacay et al., Experimentalpretargeting studies of cancer with a humanized anti-CEA x murineanti-[In-DTPA] bispecific antibody construct and a(99m)Tc-/(188)Re-labeled peptide, Bioconjug. Chem. 11:842-854, 2000.

The radiolabeling of Ac-Phe-Lys(-DTPA)-Tyr-Lys(-DTPA)-NH₂ with ¹¹¹In isdescribed in Boerman et al., Pretargeting of renal cell carcinoma:improved tumor targeting with a bivalent chelate, Cancer Res.59:4400-4405, 1999.

SUMMARY OF THE INVENTION

The present invention provides multimeric targetable complexes that aremultivalent and/or polyspecific. The invention further provides methodsof making such complexes, compositions for making such complexes, andmethods of using the multimeric targetable complexes of the invention.

Targetable Complexes

The present invention relates to multimeric targetable complexes thatare multivalent and/or polyspecific. A non-limiting example of amultimeric, multivalent targetable complex is a tetravalent targetablecomplex, which comprises (a) a targetable construct and (b) 2 moleculesof a bi-specific antibody, each molecule comprising (i) two arms, eachof which binds a carrier epitope, and (ii) two arms, each of which iscapable of binding a target epitope. The complex is tetravalent becauseit comprises a total of 4 arms, each of which is capable of binding atarget epitope (2 molecules of an antibody that has 2 arms capable ofbinding the target epitope).

A targetable complex of the invention may be polyspecific, multivalent,or both.

A tetravalent complex is examplary of a multivalent complex and may be ahomodimer or a heterodimer.

In a homodimer (e.g., a tetravalent targetable complex of the invention)both of 2 bi-specific antibodies have 2 arms that bind the same targetepitope, and 2 arms that bind a carrier epitope. The homodimeric complexis bi-specific because its arms either recognize (a) a carrier epitopeor (b) a target epitope, and is tetravalent because it has 4 arms thatrecognize the target epitope.

A tetravalent targetable complex of the invention has four arms capableof binding a target epitope, and comprises:

-   -   (a) a targetable construct comprising (i) a molecular scaffold        and (ii) two pairs of a carrier epitope; and    -   (b) two molecules of a bi-specific antibody, each antibody        comprising (i) two arms, each arm comprising a binding site for        said carrier epitope, and (ii) two arms, each comprising a        binding site for said target epitope.

Preferably, a tetravalent complex of the invention has one or more ofthe following attributes:

(I) the targetable complex has a Kd for said target epitope from about0.1 nM to about 100 nM,

-   -   (II) mixing the targetable construct and the bi-specific        antibody at relative concentrations ranging from about 10⁻³ to        about 10³ results in a mixture in which greater than about 75%        of the complexes therein have a defined stoichiometry of two        molecules of said bi-specific antibody, and one molecule of said        targetable construct, and    -   (III) a pair of carrier epitopes is bound by said bi-specific        antibody in a 1:1 ratio.

In a heterodimer (e.g., a targetable complex that is divalent for eachof two target epitopes), each of 2 bi-specific antibodies have two armsthat bind different target epitopes, and two arms that bind a carrierepitope. The heterodimeric complex is bi-specific (it recognizes twodifferent target epitopes) and divalent for each target epitope becauseit has four arms, wherein two of said arms recognize a first carrierepitope, and wherein the other two arms recognize a second targetepitope.

A targetable complex of the invention that is divalent for each of twotarget epitopes comprises:

-   -   (a) a targetable construct comprising (i) a molecular scaffold        and (ii) two pairs of carrier epitopes, wherein the first of        said two pairs of carrier epitopes is specifically bound by a        first bi-specific antibody, and the second of said two pairs of        carrier epitopes is specifically bound by a second bi-specific        antibody, wherein the targetable construct forms a targetable        complex when combined with    -   (b) a first bi-specific antibody, the first bi-specific antibody        comprising (i) two copies of a first arm comprising a binding        site for said carrier epitope, and (ii) two copies of a second        arm comprising a binding site for a first target epitope, and    -   (c) a second bi-specific antibody, the second bi-specific        antibody comprising (i) two copies of a first arm comprising a        binding site for said carrier epitope, and (ii) two copies of a        second arm comprising a binding site for a second target        epitope.

Preferably, a targetable complex of the invention that is divalent foreach of two target epitopes has one or more of the following attributes:

-   -   (I) said targetable complexes have a Kd for said target epitope        from about 0.1 nM to about 100 nM,    -   (II) mixing said targetable construct and said bi-specific        antibody at relative concentrations ranging from about 10⁻³ to        about 10³ results in a mixture in which greater than about 75%        of the complexes therein have a defined stoichiometry of two        molecules of said bi-specific antibody, and one molecule of said        targetable construct, and    -   (III) each pair of carrier epitopes is bound by one of said        bi-specific antibodies in a 1:1 ratio.

Both homodimeric and heterodimeric tetrameric complexes are encompassedby targetable complexes that comprise:

-   -   (a) a targetable construct comprising (i) a molecular scaffold        and (ii) two pairs of carrier epitopes, wherein the first of        said two pairs of carrier epitopes is specifically bound by a        first bi-specific antibody, and the second of said two pairs of        carrier epitopes is specifically bound by a second bi-specific        antibody, wherein said targetable construct forms a targetable        complex when combined with    -   (b) a first bi-specific antibody, said first bi-specific        antibody comprising (i) two copies of a first arm comprising a        binding site for said carrier epitope, and (ii) two copies of a        second arm comprising a binding site for a first target epitope,        and    -   (c) a second bi-specific antibody, said second bi-specific        antibody comprising (i) two copies of a first arm comprising a        binding site for said carrier epitope, and (ii) two copies of a        second arm comprising a binding site for a second target        epitope;    -   wherein said first bi-specific antibody and said second        bi-specific antibody can be the same or different, said pairs of        carrier epitopes can be the same or different, and said target        epitopes can be the same or different, and wherein one or more        of the following applies:    -   (I) the targetable complex has a Kd for a target epitope from        about 0.1 nM to about 100 nM,    -   (II) mixing the targetable construct and the bi-specific        antibody at relative concentrations ranging from about 10⁻³ to        about 10³ results in a mixture in which greater than about 75%        of the complexes therein have a defined stoichiometry of two        molecules of the bi-specific antibody, and one molecule of the        targetable construct, and    -   (III) each pair of carrier epitopes is bound by one of said        bi-specific antibodies in a 1:1 ratio.

In general, multivalent and/or polyspecific complexes comprise:

-   -   a targetable construct comprising a molecular scaffold and X        pairs of carrier epitopes, wherein each of said pairs of carrier        epitopes is specifically bound by one of X bi-specific        antibodies, each bi-specific antibody comprising (a) two copies        of a first arm comprising a binding site for a carrier epitope,        and (b) two copies of a second arm comprising a binding site for        one of Y target epitopes, wherein    -   (i) X is a whole integer ≧2,    -   (ii) Y is a whole integer >1,    -   (iii) said X bi-specific antibodies can be the same or a mixture        of different bi-specific antibodies,    -   (iv) said X pairs of carrier epitopes can be the same or a        mixture of different carrier epitopes, and    -   (v) when Y>2, said Y target epitopes can be the same or a        mixture of different target epitopes, and    -   a pair of carrier epitopes is bound by a bi-specific antibody in        a 1:1 ratio.        Targetable Constructs

The present invention relates to multivalent targetable constructs thatmay be used to form targetable complexes with bi-specific antibodiesprior to and/or after administration to a subject. The constructscomprise two or more pairs of carrier epitopes that are bound by an armof a bi-specific antibody and are thus multivalent constructs.

For tetrameric complexes, a tetravalent (i.e., comprising two pairs ofcarrier eptiopes) targetable construct comprises a molecular scaffold,and two pairs of a carrier epitope, wherein the targetable construct,when combined with a bi-specific antibody comprising (i) two copies of afirst arm comprising a binding site for the carrier epitope, and (ii)two copies of a second arm comprising a binding site for a targetepitope, forms a targetable complex.

A tetrameric targetable complex of the invention that is divalent foreach of two target epitopes comprises a molecular scaffold and two pairsof carrier epitopes, wherein the first pair of carrier epitopes isspecifically bound by a first bi-specific antibody, and the second pairof carrier epitopes is specifically bound by a second bi-specificantibody, wherein the targetable construct forms a targetable complexwhen combined with

-   -   (a) a first bi-specific antibody, the first bi-specific antibody        comprising (i) two copies of a first arm comprising a binding        site for a carrier epitope, and (ii) two copies of a second arm        comprising a binding site for a first target epitope, and    -   (b) a second bi-specific antibody, said second bi-specific        antibody comprising (i) two copies of a first arm comprising a        binding site for said carrier epitope, and (ii) two copies of a        second arm comprising a binding site for a second target        epitope.

In general, multivalent and/or polyspecific constructs comprise amolecular scaffold and X pairs of carrier epitopes, wherein each of saidpairs of carrier epitopes is specifically bound by one of X bi-specificantibodies, each bi-specific antibody comprising (a) two copies of afirst arm comprising a binding site for a carrier epitope, and (b) twocopies of a second arm comprising a binding site for one of Y targetepitopes, wherein

-   -   (i) X is a whole integer >2,    -   (ii) Y is a whole integer >1,    -   (iii) said X bi-specific antibodies can be the same or a mixture        of different bi-specific antibodies,    -   (iv) said X pairs of carrier epitopes can be the same or a        mixture of different carrier epitopes, and    -   (v) when Y>2, said Y target epitopes can be the same or a        mixture of different target epitopes, and    -   a pair of carrier epitopes is bound by a bi-specific antibody in        a 1:1 ratio.

Preferably, a targetable complex formed using a targetable construct ofthe invention of the invention has one or more of the followingattributes:

-   -   (a) the targetable complex has a Kd for the target epitope from        about 0.1 nM to about 100 nM, and    -   (b) mixing the targetable construct and the bi-specific antibody        at relative concentrations ranging from about 10⁻³ to about 10³        results in a mixture in which greater than about 75% of the        complexes therein have a defined stoichiometry of two molecules        of the bi-specific antibody, and one molecule of the targetable        construct,    -   (c) a pair of carrier epitopes is simultaneously bound by said        two copies of a first arm comprising a binding site for said        carrier epitope, wherein said two copies of a first arm        comprising a binding site for said carrier epitope are part of        said bi-specific antibody.        Embodiments

It has been discovered that it is advantageous to prepare and usemultimers of polyspecific antibodies, e.g., antibodies that have two ormore arms that specifically bind a targetable construct that is capableof carrying one or more therapeutic agents, and two or more arms thatbind a targeted tissue. By utilizing this technique, the multimerizationof bi-specific antibodies and other polyspecific antibodies, targetableconstructs, chelators, metal chelate complexes, therapeutic agents canbe varied to accommodate differing applications.

Because a polyspecific antibody must, by definition, specifically bindat least two different targets, a bispecific antibody (bsAb) is thesimplest type of polyspecific antibody. Similarly, a multivalentantibody must, by definition, have at least two binding sites for asingle target, and a divalent antibody is thus the simplest type ofmultivalent Abs. Thus, at a minimum, the Abs of the invention arebispecific (i.e., bind 2 different targets) and divalent (comprising 2copies of an arm that binds a target).

An antibody used in in the invention may be a monoclonal antibody, ahumanized antibody, a human antibody, a chimeric antibody, a singlechain antibody, a camelid antibody, a CDR, a soluble TCR, a fusionprotein, a naked antibody, or a fragment of any of the preceding.

The targetable construct may comprise a peptide; a carbohydrate; and/orone or more haptens including but not limited to a chelator ormetal-chelate complex. By way of non-limiting example, the chelator maybe a hard base chelator for a hard acid cation, and at least one of thechelators is a soft base chelator for a soft acid cation; or a hard basechelator that comprises carboxylate and amine groups. Non-limitingexamples of hard base chelators include DTPA, NOTA, DOTA and TETA.

The invention further relates to methods utilizing multivalent and/orpolyspecific targetable constructs and complexes. In some embodiments,the targetable construct or complex comprises a biologically activemoiety, such as one that initiates, enhances, limits or prevents abiochemical process. A bioactive moiety of the present invention isselected from the group consisting of a drug, a prodrug, an enzyme, ahormone, an immunomodulator, an oligonucleotide; a radionuclide, animage enhancing agent and a toxin. A “biochemical process” is anyprocess that alters any activity or process of a cell, or of asubcellular portion. A subcellular portion may be an organelle, e.g., amitochondrion, the endoplasmic reticulum, the nucleus, the nucleolus, orthe cell membrane and/or a receptor thereon. By way of non-limitingexample, a biochemical process may be a signaling cascade, a complementcascade, apoptosis, a biochemical pathway, or one or more reactions thatoccur in any of the preceding.

A biochemical process comprises one or more reactions. A “reaction” isany response of one or more molecules to being brought into contact withone or more other molecules, or any response of one or molecules to achange in the proximity of one or more other molecules. A chemicalreaction, in which a molecule is split into two or more molecules,and/or two or more molecules are reacted with each other to form one ormore different molecules, is a non-limiting example of a reaction. Anon-covalent association of two or more molecules with each other isanother example of a reaction. A transfer of an electron or ion from onemolecule to another is another example of reaction. A change inconformation in response to contact with another molecule is anotherexample of a reaction. Intracellular and intercellular translocations ofmolecules are reactions.

In some embodiments, the targetable constructs and complexes arebiologically active not because they comprise a bioactive moiety per sebut because the binding of the targetable construct or complex to itstargeted tissue initiates, enhances, limits or prevents a biochemicalprocess. For example, in some embodiments, the bi-specific antibodies ofthe targetable complex may be naked antibodies. A “naked antibody” is,generally, an antibody that lacks the Fc portion of an antibody. The Fcportion of the antibody molecule provides effector functions, such ascomplement fixation and ADCC (antibody dependent cell cytotoxicity),which set mechanisms into action that may result in cell lysis. However,the Fc portion may nor be required for therapeutic function in everyinstance, with other mechanisms, such as apoptosis, coming into play.

The targetable constructs and complexes may comprise one or more agentsuseful for killing or slowing the growth of diseased tissue. By way ofnon-limiting example, the agent may be a radioactive isotope,particularly the therapeutically useful therapeutic radionuclides setforth herein. The agent may also be a toxin; one or more drugs; and/orone or more prodrugs. By way of non-limiting example, the targetableconstruct or complex may comprise doxorubicin, CPT-11 or SN38.

One embodiment of the invention involves using compositions and methodsof the disclosure in boron neutron capture therapy (BNCT). In BNCT, thetargetable constructs comprise boron atoms, in which case the methodfurther comprise the step of irradiating the boron atoms localized atthe diseased tissue, thereby effecting BNCT.

Various embodiments of the invention provide pre-targeting methods andcompositions using pre-formed targetable complexes of the invention. Atargetable complex comprises a multivalent targetable construct, whichoptionally carries one or more bioactive agents; and one or more pairsof a bi-specific antibody comprising a pair of arms that specificallybind the multivalent targetable construct, and two or more arms thatbind a targeted tissue.

A further embodiment of the invention involves a kit comprising thetargetable complexes of the invention, which may further comprise one ormore compounds selected from the group consisting of one or moreradioactive isotopes useful for killing or slowing the growth ofdiseased tissue, one or more toxins, one or more drugs, and one or moreprodrugs.

In a further embodiment, the invention provides compositions and methodsfor targeting cardiovascular lesions such as atherosclerotic plaques,vascular clots including thrombi and emboli, myocardial infarction, andother organ infarcts.

The invention also provides compositions and methods for targetingmetabolic disease, such as amyloid in amyloidosis, as well as aneurodegenerative disease such as Alzheimer's disease.

In a further embodiment, the invention provides compositions and methodsfor treating a mammal having a hypoplastic, absent, anatomicallydisplaced or ectopic tissue or organ.

In a further embodiment, the invention provides compositions and methodsfor treating diseases, including, for example, pathogenic diseases,cancer, cardiovascular diseases, neurodegenerative diseases, metabolicdiseases, and autoimmune diseases.

In a further embodiment, the invention provides pharmaceuticalcompositions and kits comprising the compositions of the invention.

In a further embodiment, the invention provides compositions and methodsfor making a biosensor that may be used to detect substances in samplesor in the environment.

In a further embodiment, the invention provides compositions and methodsfor in vitro immunochemical methods, including but not limited toimmunoassays and immunoaffinity purification. In these and otherembodiments, the compositions of the invention may be attached to solidsupports. Representative solid supports include dipsticks, beads,multititer plates, the interior surface of wells in amultiwell/microtiter plate, and membranes.

In a further embodiment, the invention provides compositions and methodsfor separating a compound of interest from undesirable substances in acomposition. In this embodiment, a targetable construct or complex isattached to a solid support, to which the composition is contacted. Acompound of interest or an undesirable substance is bound by thetargetable construct or complex that is attached to the solid support.In a further step, the compound of interest or the undesirable substancesubstance are separated from each other as either the compound ofinterest or the undesirable substance is retained by the immobilizedtargetable construct or complex. The compositions and methods of theinvention can be used in a dialysis machine or system, as well as in amanufacturing process.

In addition, the present invention provides a bi-specific antibodyhaving the structure [IgG₁]-[scFv]2; where the antibody has a pair ofheavy chains and a pair of light chains, where each heavy chain has anIgG1 heavy chain and an scFv, and where the scFv is fused to theC-terminus of the IgG1 heavy chain, optionally via a linker peptide. Theantibody binding sites formed by the heavy chain and the light chain mayspecifically bind to an epitope on a targeted tissue. Each of the scFvmoieties may specifically bind to a carrier epitope. The IgG1 and/or thescFv molecules may be human, humanized, chimeric, or CDR-grafted. Theantibody further contain a bioactive moiety. The bioactive moiety maybe, for example, a drug, a prodrug, an enzyme, a hormone, animmunomodulator, an oligonucleotide; a radionuclide, an image enhancingagent and/or a toxin. The bi-specific antibody may be formulated into apharmeceutical composition.

Specific examples of such bi-specific antibodies include, but are notlimited to [hMN14-IgG1]-[734scFv]₂ and[hMN14-IgG1^((1253A))]-[734scFv]₂, [hMN14-IgG1]-[679scFv]₂ and[hMN14-IgG1^((1253A))]-[679scFv]₂, [hA20-IgG1]-[734scFv]₂ and[hA20-IgG1^((1253A))]-[734scFv]₂, [hA20-IgG1]-[679scFv]₂ and[hA20-IgG1^((1253A))]-[679scFv]₂, [hLL2-IgG1]-[734scFv]₂ and[hLL2-IgG1^((1253A))]-[734scFv]₂, and [hLL2-IgG1]-[679scFv]₂ and[hLL2-IgG1^((1253A))]-[670scFv]₂.

The invention further provides a binding complex having a tetravalentbinding molecule bound to a targetable construct, where the tetravalentbinding molecule has two binding sites for a carrier epitope and twobinding sites for a target epitope, and where the targetable constructhas a molecular scaffold and at least two carrier epitopes. Thetargetable construct may have at least two pairs of carrier epitopes andin the complex at least two of the tetravalent binding molecules may bebound to the targetable construct. The targetable construct may containat least two pairs of different carrier epitopes and in the complex thefirst tetravalent binding molecule may be bound to one pair of carrierepitopes and a second tetravalent binding molecule may be bound to asecond pair of carrier epitopes. The pairs of carrier epitopes may bedifferent epitopes. The first and second tetravalent binding moleculesmay bind to the same target epitope. The targetable construct may beselected from the group consisting of IMP 246, IMP 156, IMP 192 and IMP222. The carrier epitope may be a hapten. The carrier epitope may be achelator, where the chelator optionally is bound to a metal ion. Thechelator may be, for example, DTPA, DOTA, benzyl DTPA, NOTA, or TETA.The tetravalent binding molecule may be a bi-specific antibody havingthe structure [IgG₁]-[scFv]2, where the antibody has a pair of heavychains and a pair of light chains, and where each heavy chain has anIgG1 heavy chain and an scFv, where the scFv is fused to the C-terminusof the IgG1 heavy chain, optionally via a linker peptide. In the bindingcomplex the molecular scaffold may be, for example, a peptide or peptidederivative.

In each of these examples, the target epitope may be an antigenassociated with, for example, a disease, such as a hyperproliferativedisease, pathogenic disease, cancer, cardiovascular disease,neurodegenerative disease, metabolic disease, or autoimmune disease. Thecancer may be, for example, a cancer such as acute lymphoblasticleukemia, acute myelogenous leukemia, biliary cancer, breast cancer,cervical cancer, chronic lymphocytic leukemia, chronic myelogenousleukemia, colorectal cancer, endometrial cancer, esophageal, gastric,head and neck cancer, Hodgkin's lymphoma, lung cancer, medullarythyroid, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer,glioma, melanoma, liver cancer, prostate cancer, and/or urinary bladdercancer. The target epitope may be, for example, a tumor associatedantigen selected from the group consisting of A3, antigen specific forA33 antibody, BrE3, CD1, CD1a, CD3, CD5, CD15, CD19, CD20, CD21, CD22,CD23, CD25, CD30, CD45, CD74, CD79a, CD80, HLA-DR, NCA 95, NCA90, HCGand its subunits, CEA, CSAp, EGFR, EGP-1, EGP-2, Ep-CAM, Ba 733,HER2/neu, KC4, KS-1, KS1-4, Le-Y, MAGE, MUC1, MUC2, MUC3, MUC4, PAM-4,PSA, PSMA, RS5, S100, TAG-72, p53, tenascin, IL-6, insulin growthfactor-1 (IGF-1), Tn antigen, Thomson-Friedenreich antigens, tumornecrosis antigens, VEGF, 17-1A, an angiogenesis marker, a cytokine, animmunomodulator, an oncogene marker, an oncogene product, and othertumor associated antigens.

The present invention also provides a method of treating a disease in asubject, by administering to a subject suffering from the disease (i) atetravalent binding molecule having two binding sites for a carrierepitope and two binding sites for a target epitope, where the targetepitope is an epitope associated with the disease, (ii) optionally, aclearing agent, and (iii) a targetable construct having a molecularscaffold and at least two carrier epitopes. The disease may be, forexample, a hyperproliferative disease, pathogenic disease, cancer,cardiovascular disease, neurodegenerative disease, metabolic disease, orautoimmune disease. The targetable construct used in the method maycontain a bioactive moiety.

The present invention also provides a method of diagnosing/detecting adisease in a subject, by administering to a subject suspected ofsuffering from the disease (i) a tetravalent binding molecule having twobinding sites for a carrier epitope and two binding sites for a targetepitope, (ii) optionally, a clearing agent, and (iii) a targetableconstruct having a molecular scaffold and at least two carrier epitopes,where the construct has a detectable label. The target epitope may, forexample, be contained within, displayed by or released from one or morecells, tissues, organs or systems of the subject.

The present invention also provides a kit, having (i) a tetravalentbinding molecule having two binding sites for a carrier epitope and twobinding sites for a target epitope, (ii) optionally, a clearing agent,and (iii) a targetable construct having a molecular scaffold and atleast two carrier epitopes.

Additional aspects, features and advantages of the invention will be setforth in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theembodiments and advantages of the invention may be realized and obtainedby means of the instrumentalities and combinations particularly pointedout in the appended embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates hMN14-734scFv, an example of abispecific and divalent antibody of the invention. See published PCTApplication WO 99/66951 for details of the structure and prepartion ofhMN14-734scFv. Symbols: solid black rectangle, molecular scaffold;→carrier epitopes, open circle, optional biologically active moiety;filled diamonds, target epitopes; and ovals, antibody domains. Each pairof filled and open ovals (bottom) represent an arm of the bi-specificantibody that binds a carrier epitope, and each pair of striped andcross-hatched ovals (top) represent an arm of the bi-specific that bindsa target epitope.

FIG. 2 schematically illustrates a tetravalent targetable complex of theinvention. Two bi-specific and divalent antibody molecules are shownbound to a targetable construct. Symbols are the same as in FIG. 1.

FIGS. 3-7 show HPLC traces of peptide/antibody complexes.

FIG. 8 shows HPLC traces of targetable complexes formed by mixing IMP246 with hMN-14 IgG^((1253A))(734scFV)₂ (panel A) or with m734 IgG(panel B) mixed in a ratio of 1:10 (peptide:antibody).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides targetable complexes comprisingmultivalent and polyspecific (e.g., bi-specific) antibodies. Bi-specificantibodies have at least one arm that specifically binds a targetedtissue and at least one other arm that specifically binds a targetableconstruct.

A “targeted tissue” is a system, organ, tissue, cell, intracellularcomponent, receptor, or organelle to which a targetable construct ispreferentially delivered. In the therapeutic aspects of the invention,the targeted tissue is infected and/or misfunctioning (e.g., cancercells, infected cells, ectopic cells, etc.).

In addition to antibodies, the complexes comprise at least onetargetable construct. A “targetable construct” comprises a molecularscaffold which comprises or bears at least two pairs of carrier epitopesrecognized by the arm of the bi-specific antibody. As used herein, a“molecular scaffold” (or simply “scaffold”) is any chemical structure towhich epitopes and other moieties can be attached at a variety ofpositions, and/or with a variety of orientations, relative to thescaffold and/or other moieties. Non-limiting examples of molecularscaffolds include polymers such as peptides or peptide derivatives,oligopeptides and oligonucleotides. See Skerra, Engineered proteinscaffolds for molecular recognition, J. Mol. Recog. 13:167-187, 2000;Erratum in: J. Mol. Recog. 14:141, 2001. The oligonucleotides can beantisense oligonucleotide molecules or genes that correspond to p53.Also, an oligonucleotide, such as an antisense molecule inhibiting bcl-2expression is described in U.S. Pat. No. 5,734,033 (Reed).

The epitopes of a targetable construct are called “carrier eptiopes”herein. As used herein, the term “epitope” (also known as “immunogenicrecognition moiety”) encompasses any molecule or moiety that isspecifically bound by a recognition moiety or molecule. Non-limitingexamples of recognition moieties and molecules include antibodies,antibody derivatives, antigen-binding regions and minimal recognitionunits of antibodies, and receptor-specific ligands.

Non-limiting examples of recognizable haptens include, but are notlimited to, chelators, such as diethylenetriaminepentaacetic acid(DTPA), histamine-succinyl glycine (HSG), fluorescein isothiocyanate(FITC), vitamin B-12 and other moieties to which specific antibodies canbe raised, with scFv being preferred. Antibodies raised to the HSG orDTPA hapten are known and the scFv portion of the antibody can be usedas a carrier epitope binding arm of a bi-specific antibody. Binding ofthe carrier eptiopes is highly specific for each scFv component.

The targetable construct is multivalent, with bivalent peptides beingthe preferred peptide. The targetable construct may, but need not, belinked or conjugated to a variety of agents useful for treatment.Alternatively, the targetably construct may be administered incombination with such agents. Examples of such agents include, but arenot limited to, metal chelate complexes, folate moieties, drugs, toxinsand other effector molecules, such as cytokines, lymphokines,chemokines, immunomodulators, enzymes, radiosensitizers, asparaginase,RNAse, DNAse, carboranes, receptor targeting agents and radioactivehalogens. Additionally, enzymes useful for activating a prodrug orincreasing the target-specific toxicity of a drug can be conjugated tothe carrier. Thus, the use of a bi-specific antibody which have at leastone arm that specifically binds a targetable construct allows a varietyof applications to be performed without raising new bsAb for eachapplication.

The term “antibody fragment” (also known as “antibody derivative”)encompasses any synthetic or genetically engineered protein that actslike an antibody by binding to a specific antigen to form a complex. Forexample, antibody fragments include isolated fragments, “Fv” fragments,consisting of the variable regions of the heavy and light chains,recombinant single chain polypeptide molecules in which light and heavychain variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region. Antibodies and antibodyfragments are described in more detail below.

I. Biological Activity of Targetable Constructs and Complexes

A targetable construct may be biologically active due to the activity ofthe molecular scaffold, or the construct may optionally comprise abiologically active moiety. A targetable complex may be biologicallyactive due to the activity of the targetable construct, or the complexmay optionally comprise a biologically active moiety or molecule.

I.A. Definitions

The term “biologically active” (synonymous with “bioactive”) indicatesthat a composition or compound itself has a biological effect, or thatit modifies, causes, promotes, enhances, blocks, reduces, limits theproduction or activity of, or reacts with or binds to an endogenousmolecule that has a biological effect. A “biological effect” may be butis not limited to one that stimulates or causes an immunreactiveresponse; one that impacts a biological process in an animal; one thatimpacts a biological process in a pathogen or parasite; one thatgenerates or causes to be generated a detectable signal; and the like.Biologically active compositions, complexes or compounds may be used intherapeutic, prophylactic and diagnostic methods and compositions.Biologically active compositions, complexes or compounds act to cause orstimulate a desired effect upon an animal. Non-limiting examples ofdesired effects include, for example, preventing, treating or curing adisease or condition in an animal suffering therefrom; limiting thegrowth of or killing a pathogen in an animal infected thereby;augmenting or altering the phenotype or genotype of an animal; andstimulating a prophylactic immunoreactive response in an animal.

In the context of therapeutic applications of the invention, the term“biologically active” indicates that the composition, complex orcompound has an activity that impacts an animal suffering from a diseaseor disorder in a positive sense and/or impacts a pathogen or parasite ina negative sense. Thus, a biologically active composition, complex orcompound may cause or promote a biological or biochemical activitywithin an animal that is detrimental to the growth and/or maintenance ofa pathogen or parasite; or of cells, tissues or organs of an animal thathave abnormal growth or biochemical characteristics, such as cancercells, or cells affected by autoimmune or inflammatory disorders.

In the context of prophylactic applications of the invention, the term“biologically active” indicates that the composition or compound inducesor stimluates an immunoreactive response. In some preferred embodiments,the immunoreactive response is designed to be prophylactic, i.e.,prevents infection by a pathogen. In other preferred embodiments, theimmunoreactive response is designed to cause the immune system of ananimal to react to the detriment of cells of an animal, such as cancercells, that have abnormal growth or biochemical characteristics. In thisapplication of the invention, compositions, complexes or compoundscomprising antigens are formulated as a vaccine.

It will be understood by those skilled in the art that a givencomposition, complex or compound may be biologically active intherapeutic, diagnostic and prophylactic applications. A composition,complex or compound that is described as being “biologically active in acell” is one that has biological activity in vitro (i.e., in a cellculture) or in vivo (i.e., in the cells of an animal). A “biologicallyactive portion” of a compound or complex is a portion thereof that isbiologically active once it is liberated from the compound or complex.It should be noted, however, that such a component may also bebiologically active in the context of the compound or complex.

In order to achieve a biological effect, invention constructs maycomprise an additional moiety to facilitate internalization and/oruptake by a target cell. For aspects of the present invention thatinvolve an internalization moiety, internalization can be accomplishedin various ways. In particular embodiments, the internalization moietybinds to a recycling receptor, such as a folate receptor. For binding toa folate receptor, the internalization moiety can, for example, includefolate or methotrexate, or a folate analog binding to a folate receptor.In other embodiments, the internalization moiety includes a peptide thatenhances non-receptor mediated internalization.

Likewise, a number of internalization mechanisms can be utilized inplace of the folate receptor with folate or methotrexate. For example,hormone or hormone analog/hormone receptor pairs such as steroidhormones; specific peptide/peptide receptor; and non-receptor mediatedpeptide internalization.

A variety of different species that enhance internalization are knownand can be utilized. Examples include folic acid (folate) ormethotrexate with internalization via folate receptor; steroid hormonesand their respective receptors; receptor-recognized peptides, e.g.,somatostatin, LHRH bombesin/CCKB, substance P, VIP. In addition,antibodies that cross-link the targetable construct to a rapidlyinternalizing membrane protein can also be used to enhanceinternalization.

I.B. Therapy of Tissues and Organs

I.B.1. Cardiovascular Lesions, Atherosclerotic Plaques and VascularClots

When there is an insult to vascular endothelium, circulating bloodcells, particularly leukocytes, accumulate. Granulocytes tend toconcentrate in the largest numbers, but monocytes and lymphocytes alsoaccumulate to a lesser degree. These cells wander through the vascularendothelium to congregate in the areas of injury. The granulocytessurvive in the extravascular space for up to about three days, afterwhich the mononuclear cells, monocytes and lymphocytes, become thedominant population.

Two phases are associated with a vascular insult. The first phaseinvolves a brief early increase in vascular permeability. The moreprolonged second phase involves increased permeability, attachment ofleukocytes, mainly granulocytes, to the vessel wall, diapedesis ofpredominately leukocytes through the vessel wall, accumulation ofleukocytes in the injured area, leukocyte phagocytosis, leakage offibrinogen and platelets from the vessel, fibrin deposition in theinjured area, intravascular clotting with vessel destruction, macrophageengulfment of necrotic debris, migration of fibroblasts and formation ofconnective tissue, and the neovascularization by ingrowth ofcapillaries. Infiltration by leukocytes, particularly granulocytes, is arelatively early and significant event in the response to vascularinsult.

The well-developed atherosclerotic plaque is a result of the interplayof inflammatory and repair events, resulting in a lesion consisting ofextracellular calcium salts, cholesterol crystals, glycosaminoglycans,and blood cells and plasma components. Endothelial permeability ofarterial walls is induced in early stages of atherosclerosis, allowingthe afflux of circulating macromolecules and blood cells, particularlyleukocytes (and mainly granulocytes). Secondary changes may involvereduction in permeability of the arterial intima, and the laterdeposition of platelets and/or fibrin, proliferative, degenerative,necrotic, and repair processes that result in atheromatous lesions. Hereagain, an early component is the concentration and extravasation ofleukocytes in the injured area.

With regard to clots, when vessels are injured, plugging may occur bythe formation of fibrin, the aggregation of platelets, and combinationsof both. During these events, leukocyte sticking and aggregation,independent of platelet aggregation, occurs. Very early, even beforefibrin formation, extravasation of leukocytes takes place.

Deep vein thrombosis (DVT) and pulmonary embolism are very common in thegeneral population, affecting 30% to 60% of otherwise healthy men andwomen, and up to 80% in high-risk patients. It has been estimated thatas much as 20% of all hospital patients are affected with thromboembolicevents. In the U.S. alone, it has been estimated that 2.5 million casesoccur each year (Sherry, Sem. Nucl. Med. 7: 205-211, 1977).

The majority of commonly used nuclear medicine tests for deep veinthrombosis (DVT) involve nonspecific radiopharmaceuticals employed forradionuclide venography. There is thus an ongoing need for athrombosis-specific radiopharmaceutical for specific, sensitive, andrapid disclosure of thrombi by non-invasive external scintigraphy.Contrast venography, a common radiological method, has been the “goldstandard” for DVT, but it has a high incidence of side effects whichlimit its repeated use (Rabinov and Paulin, Arch. Surg. 104:134-144,1972). Compression B-mode ultrasound is also of use for diagnosing thepresence of thrombi in the legs, but this is region-limited and, again,not lesion-specific (Lensing et al., N. Engl. J. Med. 320:342-345,1989). Hence, radiopharmaceuticals are being sought to achievesimplicity, rapidity, and specificity for the detection and diagnosis ofDVT.

Where the aforementioned imaging agents may be useful for DVT, they mayfail to disclose pulmonary emboli, which are life-threatening lesions.Different thrombi may require different agents. Venous thrombi consistprimarily of polymers of fibrin with entrapped cells, alternating withlayers of platelets, whereas arterial thrombi are made up primarily ofaggregated platelets with less fibrin (Freiman, in: Coleman et al., eds,Hemostasis and Thrombosis—Basic Principles and Clinical Practice. NewYork, N.Y., Lippincott, 56: 766-780, 1982).

For the most part, the agents available appear to be restricted toeither fibrin-directed or platelet-directed pharmaceuticals, as reviewedby Knight, Sem. Nucl. Med. 20:52-67, 1990. Fibrin-specificradiopharmaceuticals include radiolabeled fibrinogen, soluble fibrin,antifibrin antibodies and antibody fragments, fragment E₁ (a 60 kDafragment of human fibrin made by controlled plasmin digestion ofcrosslinked fibrin), plasmin (an enzyme in the blood responsible fordissolution of fresh thrombi), plasminogen activators (e.g., urokinase,streptokinase and tissue plasminogen activator), heparin, andfibronectin (an adhesive plasma glycoprotein of 450 kDa).

Platelet-directed pharmaceuticals include radiolabeled platelets,antiplatelet antibodies and antibody fragments,anti-activated-platelets, and anti-activated-platelet factors, whichhave been reviewed by Knight (Id.), as well as by Koblik et al., Sem.Nucl. Med. 19:221-237 1989, all of which are included herein byreference. Platelet imaging is most useful during the acute phase ofthrombosis, when active platelet aggregation occurs, so that theseplatelet-based imaging methods have difficulty in disclosing clots thatare older than about 24 to about 48 hours (Oster et al., Proc. Natl.Acad. Sci. USA 82:3465-3468, 1985). Another concern is that plateletimaging may be inhibited by concurrent heparin administration in thetreatment of these patients (Seabold et al., J. Nucl. Med. 29:1169-1180,1988). Heparinization can also reduce the total number of lesions foundwith anti-fibrin antibodies (Alavi et al., J Nucl. Med. 29:825, 1988).In comparison to antifibrin antibodies, fragment El that is radiolabeledappears to demonstrate clots earlier (Koblik et al., supra). However,the fragment E₁ is difficult to isolate and prepare, and its binding toblood clots is transient (Knight et al, Radiology 156:509-514, 1985).

Inadequate blood and oxygen supply to the myocardium, inducing symptomsof myocardial ischemia or ischemic heart disease, are the usual eventsresulting from stenotic coronary atherosclerosis. Acute and totalcoronary artery occlusion results in severe ischemia and, consequently,myocardial infarction. Chronologically, in the first hour, subcellularchanges of ischemic heart muscle manifest as mitochondrial granules,reduction of glycogen and respiratory enzymes. Thereafter, from about 1to about 6 hours, margination and clumping of nuclear chromatin, loss ofnuclear and myofilament architecture, and infiltration withgranulocytes, are observed. In the next phase, from about 6 to 12 hours,typical ischemic necrosis is seen. After 24 hours, severe histologicalchanges are easily seen, leading to focal hemorrhage of different sizeand dilated capillaries by days 2-4.

Accordingly, the present invention provides compositions and methods forthe detection and/or treatment of cardiovascular disorders, includingfibrin clots, deep vein thrombosis, emboli, ischemia, andatherosclerotic plaques.

I.B.2. Anatomically Displaced or Ectopic Tissues and Organs

The invention provides compositions and methods for treating a mammalhaving a hypoplastic, absent, anatomically displaced or ectopic tissueor organ. Where normal organs or tissues are developed abnormally or aredisplaced in the body, or are insufficiently removed during ablativesurgery, the tissue/organ-associated antibodies may be used astissue-targeted vehicles for delivering therapeutic agents to thetissues in order to induce their involution or chemical and/or isotopicablation. The antibodies or their fragments, or recognition moities, canbe conjugated to or administered in combination with therapeuticmodalities including, but not limited to, isotopes, drugs, toxins,photodynamic therapy agents, cytokines, hormones, autocrines, etc.,which are used as cytotoxic or modulating agents, and which havehitherto been employed principally as toxic conjugates tocancer-targeting antibodies, as described in reviews by Waldmann,Science 252:1657, 1991; Koppel, Bioconjug. Chem. 1:13, 1990; Oeltmannand Frankel, FASEB J. 5:2334, 1991; and van den Bergh, Chemistry inBritain, May 1986, 430-439, each of which is incorporated by referenceherein in its entirety.

The method comprises the steps of (a) parenterally injecting a mammaliansubject, at a locus and by a route providing access to the tissue ororgan, with an amount of a scintigraphic imaging agent or magneticresonance image enhancing agent sufficient to permit a scintigraphicimage or an enhanced magnetic resonance image of the structure to beeffected; and (b) obtaining a scintigraphic image or an enhancedmagnetic resonance image of the structure, at a time after injection ofthe agent sufficient for the agent to accrete in the structure. Thetargetable complex comprises an antibody or antibody fragment thatspecifically binds to the organ or tissue, and further comprises abioactive (therapeutic) agent. In the case of naked antibodies, thecomplex itself may be biologically active and induce processes such asapoptosis.

Tissues, organs, and conditions of interest include but are not limitedto:

-   -   (1) hypoplastic or absent tissue or organs, in conditions such        as, juvenile diabetes, wherein the islet cells of the pancreas        can be atrophic or significantly reduced; thymic aplasia or        agenesis; DiGeorge's Syndrome wherein there is a hypoplasia or        absence of parathyroid and the thymus;    -   (2) ectopic tissue and organs, such as, implants of endometrial        glands and stroma;    -   (3) retained tissue, such as, retained placental tissue after        pregnancy, and organ remnants after surgical removal of the        organ;    -   (4) the condition of organs adjacent to a surgically removed        organ; and    -   (5) ablation of certain normal organs and tissues for other        therapeutic purposes, such as the spleen in patients with immune        disease or lymphomas, the bone marrow in patients requiring bone        marrow transplantation, or normal cell types involved in        pathological processes, such as certain T-lymphocytes in        particular immune diseases.

The above methods of the invention include the use of a growth factorreceptor antibody or a hormone receptor antibody to target to end-organsbearing such receptor(s), the functions of which can be blocked withsaid antibodies. An isotopic or drug conjugate of these antibodies canalso be used to deliver a therapeutic agent to said tissues and organs,in order to affect diseases of tissues which bear such receptors. Forexample, in endometriosis, involving ectopic endometrial tissue, thecurrent standard drug therapy involves administration of a syntheticsteroid derived from ethisterone (DANOCRINE brand of danazol), which ischemically a 17-alpha-Pregna-2,3-dien-20-yno[2,3,3-d]-isoxazol-17-ol.This probably acts, at least in part, on sex steroid metabolism and withsex hormone receptors, particularly follicle-stimulating hormone (FSH)and luteinizing hormone (LH) at the target organ. It is now possible touse an antibody against these gonadal steroid receptors, alone or as animmunoconjugate with isotopes, drugs, toxins, hormone antagonists,cytokines, autocrines, etc., to inactivate and make the ectopicendometrium atrophic.

The above methods of the invention include providing an immunologicalmethod of affecting ovarian and other hormone end-organ function, suchas to induce amenorrhea or sterility. By use of an ovarian-targetingantibody or an antibody to an ovarian-related hormone receptor, such asFSH receptor, either as unconjugated antibodies or as antibodiesconjugated with a therapeutic principle, a relatively convenient andsafe method of blocking ovarian function and inducing atrophy at theend-organ can be achieved.

Many hormone and growth factor receptors are known, and frequently showsufficient organ and tissue proclivity to allow these to serve astargets for antibodies which, when bound to said receptors, affect thefunction of the tissues and result in an immunological or, by the use ofconjugates with drugs, a chemical ablation, or a radiation ablation whenused as a conjugate with therapeutic isotopes.

Another application is in the treatment of fibrocystic breast disease.An antibody to FSH receptor or to estrogen receptor can be given aloneor as an immunoconjugate with a therapeutic principle to decrease thefibrocystic disease and to control its symptoms.

Still another indication is in benign prostatic hyperplasia or prostaticcancer, where the use of an antibody against an androgen receptor canalone, or as a conjugate with a therapeutic principle (hormone end-organantagonist, cytotoxic drug, toxin, or isotope), can decrease theprostatic tissue proliferation.

Another therapeutic application for such organ- and tissue-targetingantibodies conjugated with a toxic agent is for the ablation of certainnormal cells and tissues as part of another therapeutic strategy, suchas in bone marrow ablation with antibodies against bone marrow cells ofparticular stages of development and differentiation, and in thecytotoxic ablation of the spleen in patients with lymphoma or certainimmune diseases, such as immune thrombocytopenic purpura, etc.

Another therapeutic application for such organ- and tissue-targetingantibodies or fragments is to link them to a cytoprotective agent toform therapuetic conjugates. The conjugate is administered to a patientundergoing chemotherapy or radiation therapy so that the targeted normalorgans and tissues are protected during the therapy.

I.B.3. Cancer

The present invention further provides compositions and methods fortreating a disease state selected from the group consisting of acarcinoma, a melanoma, a sarcoma, a neuroblastoma, a leukemia, a glioma,a lymphoma and a myeloma. Specific tumor-associated antigens may beassociated with a type of cancer selected from the group consisting ofacute lymphoblastic leukemia, acute myelogenous leukemia, biliary,breast, cervical, chronic lymphocytic leukemia, chronic myelogenousleukemia, colorectal, endometrial, esophageal, gastric, head and neck,Hodgkin's lymphoma, lung, medullary thyroid, non-Hodgkin's lymphoma,ovarian, pancreatic, glioma, melanoma, liver cancer, prostate, andurinary bladder. A tumor-associated antigen may be selected from thegroup consisting of A3, the antigen specific for the A33 antibody, BrE3,CD1, CD1a, CD3, CD5, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30,CD33, CD45, CD74, CD79a, CD80, NCA90, NCA 95, HLA-DR, CEA, CSAp, EGFR,EGP-1, EGP-2, Ep-CAM, Ba 733, HER2/neu, KC4, KS-1, KS1-4, Le-Y, S100,MAGE, MUC1, MUC2, MUC3, MUC4, PAM-4, PSA, PSMA, AFP, HCG and istsubunits, RS5, TAG-72, tenascin, IL-6, insulin growth factor-1 (IGF-1),Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens,VEGF, 17-1A, an angiogenesis marker, a cytokine, an immunomodulator, anoncogene marker (e.g., p53), and an oncogene product.

Tumor-associated markers have been categorized by Herberman (see, e.g.,Immunodiagnosis of Cancer, in THE CLINICAL BIOCHEMISTRY OF CANCER,Fleisher ed., American Association of Clinical Chemists, 1979) in anumber of categories including oncofetal antigens, placental antigens,oncogenic or tumor virus associated antigens, tissue associatedantigens, organ associated antigens, ectopic hormones and normalantigens or variants thereof. Occasionally, a sub-unit of atumor-associated marker is advantageously used to raise antibodieshaving higher tumor-specificity, e.g., the beta-subunit of humanchorionic gonadotropin (HCG) or the gamma region of carcinoembryonicantigen (CEA), which stimulate the production of antibodies having agreatly reduced cross-reactivity to non-tumor substances as disclosed inU.S. Pat. Nos. 4,361,644 and 4,444,744. Markers of tumor vasculature(e.g., VEGF), of tumor necrosis, of membrane receptors (e.g., folatereceptor, EGFR), of transmembrane antigens (e.g., PSMA), and of oncogeneproducts can also serve as suitable tumor-associated targets forantibodies or antibody fragments. Markers of normal cell constituentswhich are overexpressed on tumor cells, such as B-cell complex antigens,as well as cytokines expressed by certain tumor cells (e.g., IL-2receptor in T-cell malignancies) are also suitable targets for theantibodies and antibody fragments of this invention.

The BrE3 antibody is described in Couto et al., Cancer Res.55:5973s-5977s, 1995. The EGP-1 antibody is described in U.S.Provisional Application Ser. No. 60/360,229, some of the EGP-2antibodies are cited in Staib et al., Int. J. Cancer 92:79-87, 2001; andSchwartzberg et al., Crit. Rev. Oncol. Hematol. 40:17-24, 2001. The KS-1antibody is cited in Koda et al., Anticancer Res. 21:621-627, 2001; theA33 antibody is cited in Ritter et al., Cancer Res. 61:6854-6859, 2001;Le(y) antibody B3 is described in Di Carlo et al., Oncol. Rep.8:387-392, 2001; and the A3 antibody is described in Tordsson et al.,Int. J. Cancer 87:559-568, 2000.

Also of use are antibodies against markers or products of oncogenes, orantibodies against angiogenesis factors, such as VEGF. VEGF antibodiesare described in U.S. Pat. Nos. 6,342,221, 5,965,132 and 6,004,554, andare incorporated by reference in their entirety. Antibodies againstcertain immune response modulators, such as antibodies to CD40, aredescribed in Todryk et al., J. Immunol. Meth. 248:139-147, 2001 andTurner et al., J. Immunol. 166:89-94, 2001. Other antibodies suitablefor combination therapy include anti-necrosis antibodies as described inEpstein et al., see e.g., U.S. Pat. Nos. 5,019,368; 5,882,626; and6,017,514.

I.B.4. Autoimmune Diseases

The present invention further provides compositions and methods fortreating an autoimmune disease or disorder. Immunothereapy of autoimmunedisorders using antibodies which target B-cells is described in PCTApplication Publication No. WO 00/74718, which claims priority to U.S.Provisional Application Ser. No. 60/138,284, the contents of each ofwhich is incorporated herein in its entirety. Exemplary autoimmunediseases are acute idiopathic thrombocytopenic purpura, chronicidiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetesmellitus, Henoch-Schonlein purpura, post-streptococcalnephritis,erythema nodosurn, Takayasu's arteritis, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitisubiterans, Sjogren'ssyndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia,pemiciousanemia, rapidly progressive glomerulonephritis and fibrosingalveolitis.

I.C. Therapeutic Applications

I.C.1. Photodynamic Diagnosis or Therapy (PDT)

The present mutant bsAb can be used in a method of photodynamic therapy(PDT) as discussed in U.S. Pat. Nos. 6,096,289; 4,331,647; 4,818,709;4,348,376; 4,361,544; 4,444,744; 5,851,527.

In PDT, a photosensitizer, e.g., a hematoporphyrin derivative such asdihematoporphyrin ether, is administered to a subject. Anti-tumoractivity is initiated by the use of light, e.g., 630 nm. Alternatephotosensitizers can be utilized, including those useful at longerwavelengths, where skin is less photosensitized by the sun. Examples ofsuch photosensitizers include, but are not limited to,dihematoporphyrin, benzoporphyrin monoacid ring A (BPD-MA), tinetiopurpurin (SnET2), sulfonated aluminum phthalocyanine (AISPc) andlutetium texaphyrin (Lutex).

Radionuclides useful in therapeutic agents, which substantially decay bybeta-particle emission include, but are not limited to: P-32, P-33,Sc-47, Fe-59, Cu-64, Cu-67, Se-75, As-77, Sr-89, Y-90, Mo-99, Rh-105,Pd-109, Ag-111, I-125, I-131, Pr-142, Pr-143, Pm-149, Sm-153, Th-161,Ho-166, Er-169, Lu-177, Re-186, Re-188, Re-189, Ir-194, Au-198, Au-199,Pb-211, Pb-212, and Bi-213. Maximum decay energies of usefulbeta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV, and most preferably 500-2,500 keV.

Radionuclides useful in therapeutic agents which substantially decaywith Auger-emitting particles include, but are not limited to: Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV.

Radionuclides useful in therapeutics and which substantially decay withgeneration of alpha-particles include, but are not limited to: Dy-152,At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217,Bi-213 and Fm-255. Decay energies of useful alpha-particle-emittingradionuclides are preferably 2,000-9,000 keV, more preferably3,000-8,000 keV, and most preferably 4,000-7,000 keV.

Metals useful, as complexes, as part of a photodynamic therapy procedureinclude, but are not limited to zinc, aluminum, gallium, lutetium andpalladium.

Therapeutically useful immunoconjugates can be obtained by conjugatingphotoactive agents or dyes to an antibody composite. Fluorescent andother chromogens, or dyes, such as porphyrins sensitive to visiblelight, have been used to detect and to treat lesions by directing thesuitable light to the lesion. In therapy, this has been termedphotoradiation, phototherapy, or photodynamic therapy (Jori et al.,eds., Photodynamic Therapy of Tumors and Other Diseases (LibreriaProgetto 1985); van den Bergh, Chem. Britain 22:430, 1986). Moreover,monoclonal antibodies have been coupled with photoactivated dyes forachieving phototherapy. Mew et al., J. Immunol. 130:1473, 1983; idem.,Cancer Res. 45:4380, 1985; Oseroffet al., Proc. Natl. Acad. Sci. USA83:8744, 1986; idem., Photochem. Photobiol. 46:83, 1987; Hasan et al.,Prog. Clin. Biol. Res. 288:471, 1989; Tatsuta et al., Lasers Surg. Med.9:422, 1989; Pelegrin et al., Cancer 67:2529, 1991. However, theseearlier studies did not include use of endoscopic therapy applications,especially with the use of antibody fragments or subfragments. Thus, thepresent invention contemplates the therapeutic use of immunoconjugatescomprising photoactive agents or dyes.

I.C.2. Boron Neutron Capture Therapy (BNCT)

BNCT is a binary system designed to deliver ionizing radiation to tumorcells by neutron irradiation of tumor-localized boron-10 atoms. BNCT isbased on the nuclear reaction which occurs when a stable isotope,isotopically enriched B-10 (present in 19.8% natural abundance), isirradiated with thermal neutrons to produce an alpha particle and a Li-7nucleus. These particles have a path length of about one cell diameter,resulting in high linear energy transfer. Just a few of the short-range1.7 MeV alpha particles produced in this nuclear reaction are sufficientto target the cell nucleus and destroy it. Barth et al., Cancer70:2995-3007, 1992.

Historically, BNCT was first employed for the treatment of glioblastoma(a fatal form of brain tumor) and other brain tumors at a time whentumor specific substances were almost unknown. Hatanaka et al., in BoronNeutron Capture Therapy for Tumors, pp.349-78 (Nishimura Co., 1986). Oneof the first boronated compounds employed, a sulfhydryl-containing boronsubstance called sodium borocaptate or BSH (Na₂, B₁₂H₁₁ SH), crosses theblood-brain barrier to localize in brain, and this has been theanatomical basis for neutron capture therapy of brain tumors. Clinicaltrials have been carried out, or are scheduled, for the treatment ofgliomas in Japan, the US and Europe. Barth et al., Cancer, supra.Problems with previous inorganic boron therapy methods was that theboron reached both targeted and non-target areas. Accordingly, when theboron was irradiated, healthy cells as well as cancerous cells weredestroyed.

The BNCT concept has been extended to other cancers, spurred on by thediscovery of a number of tumor-localizing substances, includingtumor-targeting monoclonal antibodies. For instance, boronated aminoacids such as p-boronophenylalanine accumulated in melanoma cells. Thepotential of using boronated monoclonal antibodies directed against cellsurface antigens, such as CEA, for BNCT of cancers has beendemonstrated. Ichihashi et al., J. Invest. Dermatol. 78:215-18, 1982;Goldenberg et al., Proc. Natl. Acad. Sci. USA 81:560-63, 1984; Mizusawaet al., Proc. Natl. Acad. Sci. USA 79:3011-14, 1982; Barth et al.,Hybridoma 5(supp. 1):543-5540, 1986; Ranadive et al., Nucl. Med. Biol.20: 663-68, 1993.

Success with BNCT of cancer requires methods for localizing a highconcentration of boron-10 at tumor sites, while leaving non-targetorgans essentially boron-free. Compositions and methods for treatingtumors in patients using pre-targeting bsAb for BNCT are described inU.S. application Ser. No. 09/205,243 and can easily be modified inaccordance with the present invention. Additionally, other elements aresuitable for neutron capture reactions. Nuclides useful in therapiesbased on neutron capture procedures include, but are not limited to:B-10, Gd-157 and U-235. Uranium, in large amounts, can be bound bynaturally occurring chelating agents such as ferritin.

II. Pre-Formed Targetable Complexes

In therapeutic embodiments of the present invention, the bi-specificantibodies may be adminstered at some time prior to administration ofthe targetable construct. However, it is also possible to mix targetableconstructs and bi-specific antibodies prior to administration, and thusto form “pre-formed” targetable complexes that are then administered toa subject. Targetable complexes are also useful in ex vivo and in vitromodalities.

II.A. Pre-Targeting Applications

In an exemplary method that does not involve pre-targeting, thetargetable construct comprises a bioactive moiety. In this case, thetargetable construct is administered following administration of thebsAb. The bioactive agent is targeted to the target site because thetargetable construct is recognized by and binds to the bsAb, which isitself bound to a targeted tissue.

In an alternative method, a targetable construct comprising a bioactiveagent is mixed with its cognate bsAb prior to administration to thepatient, thus forming a targetable complex comprising a bioactive agent.A targetable complex formed in this fashion is administered and bindsits targeted tissue, thereby effecting direct delivery of the bioactiveagent as a part of the targetable complex comprising the agent.

The latter or pre-targteing modality has several potential advantagesover methods in which the targetable constructs and bsAb are separatelyadministered. The total amount of targetable construct and bsAb thatneeds to be administered in order to be effective may be less than innon-pre-targeting modalities, particuarly if the targetable complex isrelatively stable under physiological conditions. In addition, atargetable complex according to the invention may have a higher affinityfor the targeted tissue that the targetable construct per se, therebyproviding compositions and methods for more effective delivery of thebioactive agent to the targeted tissue.

Pre-formed targetable complexes may be used in any of the compositionsand methods of the invention. One skilled in the art will be able todetermine what site of complex formation (i.e., in vitro or in situ) isappropriate for any given application.

II.B. Immunoaffinity-Based Applications

II.B.1. Immunoaffinity

Immunoaffinity is known in the art and generally involves theimmobilization of antibodies to a solid support, often in the form ofbeads, that are then packed into a column. A sample containing anantigen recognized by the antibody is passed through the column, whereinthe antigen is bound and retained by the immobilized antibodies. Theantigen is then washed off the column using any of a variety of methodsknown in the art. Depending on the particular circumstances, the antigenmay be a substance that is being purified, or the antigen can be acontaminant that is being removed. For further details and reviews, seeSpringer, Section 10.11, Immunoaffinity Chromatography, Chapter 10 in:Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds.,John Wiley and Sons, New York, 1992, pages 10-43 to 10-45); AffinityChromatography: A Practical Approach, edited by Dean P. D. G., Johnson,W. S., Middle, F. A., IRL Press,1985; Immunoaffinity Purification: BasicPrinciples and Operational Considerations, Yarmush et al., Biotech Adv.10:412-446, 1992.

Immunoaffinity comprises three general steps: adsorption, washing andelution. In the first step, absorption, a substance of interest is boundby an antibody. Absorption is accomplished by, e.g., contacting a samplecontaining the substance of interest with an antibody bound to a solidsupport matrix in a suitable medium within a column. The next step is awashing step wherein impurities present in the fluid volume of thecolumn, as well as those bound nonspecifically to the antibody, solidsupport or column walls, are removed. Washing is accomplished by passinga volume of a wash solution, e.g., buffer, such as phosphate bufferedsaline (PBS) through the column. The volume of wash solution used in thewashing step should not be so great as to result in loss of thesubstance of interest but not so limited so as not to remove impurities.In the elution step, the target molecule is removed from the column by,e.g., addition of a solvent or other solution, or change in conditionssuch as temperature or pressure, that reduces the affinity of thesubstance of interest to the antibody or the affinity of the complexformed between an antibody and molecules of the substance of interest tothe solid support. Elution of an antibody coupled to the substance ofinterest may be accomplished by either a salt gradient, to change thepH; buffered step-gradient, to change the ionic strength; or othermethods known in the art.

Elution of the target molecule may be accomplished by a number ofmethods. There are no covalent bonds involved in the interaction betweenantibody and the substance of interest. Thus, the conditions of thebuffer may be changed such that the affinity of the antibody:substancecomplex falls sufficiently to reduce the amount of effective binding toeach other or to the solid support. This may be achieved by altering thepH or the ionic strength of the buffer, or both, or by chaotropic ions,e.g., cyanates. Increased separation may be obtained by gradientelution. In the case of immunosorption, the binding of a substance ofinterest to its antibody may be so strong that more harsh elutionconditions are necessary, such as the use of buffers which are verystrongly acidic or basic. Other methods of elution include use ofchaotropic agents such as KSCN; organic solvents, e.g., ethylene glycol,DMSO, or acetonitrile; denaturing agents, e.g., 8 M urea or 6 M guanine;electrophoretic elution; pressure induced elution and metal ion elution.Preferably, the elution conditions allows for complete or mostlycomplete elution of the product after one or two column volumes havepassed through the column.

Various impurities can be present and may have an unpredictable andadverse affect on the composition as it is used in the pharmaceuticalindustry. In the case of biological samples, typical impurities areblood clots, tissue debris, hair, foreign particles, activatedcoagulation factors, denatured proteins, plasma-free hemoglobin (e.g.,irrigation fluid) added into a wound site, human viruses, antigens andantibiotics.

By way of non-limiting example, a sample may be passed through animmunoaffinity column having an immobilized antibody directed againstthe substance of interest. The immobilized antibodies react with andbind molecules of the substance of interest in the sample, therebyabsorbing them and removing them from the solution. Although thesubstance of interest is retained on the column, impurities pass throughthe column. The column can then be washed with a buffer solution toremove any impurities remaining on the column, e.g., impurities retainedby non-specific binding. The column is washed free of impurities and anysubstance bound to the column is eluted with a solvent. This process isknown as positive immunoabsorption. In negative immunoabsorption, incontrast, the substances of interest present in the crude preparationpass freely through the column while the antigenic impurities bind withantibodies and are held by the column.

II.B.2. Immobilization of Antibodies

A solid support or matrix is used to immobilize antibodies. The matrixmay possess desirable characteristics including, macroporosity,mechanical stability, ease of activation, hydrophilicity, and inertness,i.e., low nonspecific adsorption. Matrices commonly used by thoseskilled in the art include cross-linked dextran, agarose,polyacrylamide, cellulose, silica and poly(hydroxyethylmethacrylate).For immuno-adsorbents, beaded agarose is a preferred solid support bythose skilled in the art due to its high adsorptive capacity forproteins, high porosity, hydrophilicity, chemical stability, lack ofcharge and relative inertness toward nonspecific adsorption.

Antibodies may be physically adsorbed to matrices or covalently attachedto polymeric matrices containing hydroxylic or amino groups by means ofbifunctional reagents, such as those disclosed herein. Attachmenttypically requires two steps, activation of the matrix and coupling ofthe ligand to the activated matrix. Activated matrices are availablecommercially. The selection method for coupling the ligand to the matrixis dictated in part by the choice of matrix and, in part, by the choiceof antibody. Most methods commonly used to immobilize peptide orpolypeptide ligands, such as antibodies, are based on coupling of aminogroups. The polypeptide ligand must be coupled in a manner that will notinterfere with its ability to be recognized by the target molecule.Methods for activation and coupling are commonly used by those skilledin the art.

For successful use of affinity chromatography, the polymer-bound ligandmust be sufficiently distant from the polymer surface to minimize stericinterference. This is accomplished by inserting an interconnecting linkor spacer between the antibody and the matrix. The spacer may be bounddirectly to the matrix so that the antibody can be attached directly tothese spacers. Types of spacers commonly used by those skilled in theart include but are not limited to cystamine, p-aminobenzoic acid,tyramine and p-hydroxy-mercuribenzoate.

II.B.3. Beads

In embodiments wherein the solid support is a bead, the bead may be anyof a variety of types, depending upon the application. Forimmunopurification, porous beads are often used. The beads may beprepared from commercially available beads that are derivatized withamino or carboxyl groups that are available for linkage to a protein orother capture moiety using, for example, glutaraldehyde, carbodiimide,diazoto compounds, or any other suitable crosslinking reagent.

II.B.3.a. Magnetic Beads

Targetable complexes may be attached to magnetic particles viafunctional groups that coat the particles. In a purificationapplication, a sample containing an antigen comprising a target epitopebinds to the attached targetable complex, and the conjugated magneticparticle is removed from suspension by the application of a magneticfield.

Magnetic beads or particles, such as magnetic latex beads and iron oxideparticles, to which the targetable complexes of the invention may beattached, are known in the art. For example, magnetic particles aredescribed in U.S. Pat. No. 4,672,040. Coupling of capture moieties tomagnetic beads can be accomplished using known methods. For example,beads are commercially available that are derivatized with amino orcarboxyl groups that are available for linkage to a protein or othercapture moiety using, for example, glutaraldehyde, carbodiimide, diazotocompounds, or other suitable crosslinking reagent. Silanization ofmagnetically responsive particles provides one method of obtainingreactive groups on the surface of the particles (see, e.g., U.S. Pat.No. 4,672,040 for a description of silanization and silane couplingchemistry). Linking bonds can include, for example, amide, ester, ether,sulfonalmide, disulfide, azo, and others known to those of skill in theart.

Superparamagnetic particles, which can be made from a number ofsubstances such as polystyrene or iron oxide and polysaccharides, aremagnetic when placed in a magnetic field, but retain no residualmagnetism when removed from the magnetic field. This lack of residualmagnetism ensures that the particles can be repeatedly separated andresuspended without magnetically induced aggregation.

II.B.3.b. Beads for Immunoaffinity Purification

Beads may be coated with the targetable complexes of the invention foruse in immunoaffinity purification. Generally, such beads are of a size,composition and structure suitable for use in flow-through columns.Porous beads may be used. By way of non-limiting example, such beads canbe made of Sephadex®, Sepharose, agarose, glass, and polystyrene.

II.B.4. Preparation of an Immunoaffinity Column

In column immunoaffinity, a column comprising a solid support onto whichthe construct or complex is immobilized is prepared. The preparation ofthe column depends on the type of solid support used, the chemical orphysical nature of the samples to be processed through the column,reagents, e.g., washing and elution solutions, and the like.

It may be desirable to equilibrate the column before application of asample. The buffering conditions used for equilibrating the affinitycolumn in preparation for sample application will reflect the specificproperties of the interacting system being used. The nature of thebuffer used, including its pH and ionic strength, are adjusted for theparticular antibody and substance of interest. The sample comprising thesubstance of interest that is applied to the column typically containedin the same buffer used to equilibrate the column. After sampleapplication and absorption, the column is washed with the startingbuffer to remove any unbound sample and any impurities. It may also bepreferable in some instances to wash the column with buffers differentfrom the starting buffer in order to remove nonspecifically adsorbedsubstances.

II.C. Manufacturing Embodiments

In addition to being useful for purifying compounds of interest frommixtures of compounds, immunoafinity can be used to remove undesirablesubstances from mixtures in manufacturing and other applications.Exemplary undesiarble substances include, but are not limited to,contaminants, undesirable reaction products and/or catalysts includingbut not limited to enzymes, that are used during manufacturingprocesses.

The immunoaffinity aspects of the invention may also be applied tomanufacturing processes. A manufacturing process can be a “continuousprocess,” in which the substance of interest is continually produced andharvested from an ongoing manufacturing or production process. Incontrast, in a “batch” approach in manufacturing, multiple reparationsare combined and then harvested. Regardless of the type of manufacturingprocess, the compositions of the invention can be used at any of avariety of steps in the process.

A sample or manufacturing preparation may be “clarified” prior tofurther preparation in order to remove contaminants (including withoutlimitation, in chemical syntheses, reaction byproducts and unreactedcompounds) produce a sample containing only, or enriched for, thesubstance of interest. Additionally or alternatively, a substance ofinterest may be partially purified, substantially purified or purified.A substance is said to be “partially purified” when it comprises ≧50%w/w of a composition; “substantially purified” when it comprises ≧75% ofa composition, and “purified” when it comprises ≧90%, preferably ≧95%,more preferably ≧99% and most preferably ≧99.9% of a composition.Generally, clarification of a sample removes a limited number ofundesirable compounds from a preparation without changing theconcentration of the substance. In contrast, the purification of asubstance generally refers to a process by which the substance ispreferentially removed from a sample, leaving behind a variety ofcontaminants; the separated substance may be moved to a new solution inwhich its concentration is higher.

Impurities can be removed from a preparation of a substance of interestby negative or positive immunoabsorption techniques. A preparation sotreated is said to be enriched for the substance of interest, and thesubstance of interest in the preparation is said to be partiallypurified, substantially purified or purified. The substance purified inthis manner may be an antibody that specifically binds the carriereptitope of the targetable construct, or a [target epitope]:[bi-specificantibody] complex.

When the latter type of complex is prepared, the target epitope may befurther purified by treatment with agents or conditions that reduce theaffinity of the bi-specific antibody for the target epitope. Ininstances where a targetable construct comprising a carrier epitope isattached to a solid support, and a bi-specific antibody is bound to thecarrier epitope, the [target epitope]:[bi-specific antibody] complex canbe separated from the bound targetable construct by addition of anexcess amount of the carrier epitope. By way of non-limiting example, inthe case of IMP 246 bound to a solid support, a complex comprising abispecific antibody that is bound thereto may be removed from the boundIMP 246 by the addition of an excess amount of the chelatorcorresponding to the chelator moiety present on IMP 246, i.e., DTPA.

For example, a sample may be passed through an immunoaffinity columnhaving an immobilized antibody directed against the substance ofinterest. The immobilized antibodies react with and bind molecules ofthe substance of interest in the sample, thereby absorbing them andremoving them from the solution. Although the substance of interest isretained on the column, impurities pass through the column. The columncan then be washed with a buffer solution to remove any impuritiesremaining on the column, e.g., impurities retained by non-specificbinding. The column is washed free of impurities and any substance boundto the column is eluted with a solvent. This process is known aspositive immunoabsorption. In negative immunoabsorption, in contrast,the substances of interest present in the crude preparation pass freelythrough the column while the antigenic impurities bind with antibodiesand are held by the column.

II.D. Immunoassays and Other In vitro Immunochemical Methods

The targetable complexes of the present invention may be used asreagents in a variety of in vitro immunochemical methods. Immunochemicalmethods include, but are not limited to, Western blotting,immunoaffinity purification, immunoprecipitation, ELISA, dot or slotblotting, radioimmunoassay (RIA), immunohistochemical staining,immunocytochemical staining, and flow cytometry.

Such methods may, but need not in every instance, involve the attachmentof a targetable complexes to a solid support. The term “solid support”refers to a material having a solid surface to which a targetablecomplex is immobilized. By “immobilized” it is meant bound covalently,or bound by noncovalent means such as hydrophobic adsorption. By way ofnon-limiting example, a solid support may be the surface of a multiwell(microtiter) plate well, a bead, a membrane or a dipstick. Methods andmeans for covalently or noncovalently binding proteins to solid supportsare known in the art.

Suitable solid supports include, by way of illustration and notlimitation, latex, glass particles, including porous glass particles;polyacrylamide particles; agarose; SEPHADEX® (Pharmacia Fine Chemicals,Inc.); sepharose; bibulous materials such as glass or cellulose paper;plastics and polymers (e.g., in sheets, beads or microtiter wells) suchas polystyrene, polyvinyl chloride, polystyrene latex, or polyvinylidinefluoride (known as Immulon□); nylon; polymethacrylate; etc.; silicons;metals such as gold and indium; nitrocellulose nitrocellulose (e.g., inmembrane or microtiter well form); activated beads; Protein A beads;diazotized paper; and the like.

The nature of the solid surface varies depending upon the intended useor method. For assays carried out in microtiter wells, e.g., inmultiwell (microtiter) plates, the solid surface is the wall of the wellor cup. For assays using beads, the solid surface is the surface of thebead. In assays using a dipstick (i.e., a solid body made from a porousor fibrous material such as fabric or paper) the surface is the surfaceof the material from which the dipstick is made. In agglutination assaysthe solid surface may be the surface of latex or gelatin particles. Whenindividual antigens are bound to a solid surface they may be distributedhomogeneously on the surface or distributed thereon in a pattern, suchas bands so that a pattern of antigen binding may be discerned.

II.D.1. Immunoassays

The design of immunoassays is subject to a great deal of variation, andmany formats are known in the art. Immunochemical Protocols, HumanaPress, Totowa, N.J., 1998; Current Protocols in Immunology, Greene Pub.Associates and Wiley-Interscience, New York, N.Y., 1997. Protocols may,for example, use solid supports, or immunoprecipitation. Most assaysinvolve the use of labeled targetable complex or antigen. As used inthis section, an “antigen” is a substance that is or comprises atargetable epitope. The labels may be, for example, enzymatic,fluorescent, chemiluminescent, radioactive, or dye molecules. Assayswhich amplify the signals are known; examples of which are assays whichutilize biotin and avidin, enzyme-labeled and mediated immunoassays,such as ELISA, RIA, immunofluorescence, chemiluminescence andnephelometry.

Typically, standard ELISA techniques are employed using labelledantibody or antigen. The label can be an enzyme, fluorophore,chemiluminescent material, radioisotope, or coenzyme. Generally enzymelabels such as alkaline phophatase, or beta galactosidase are employedtogether with their appropriate substrates. The enzyme/substratereaction can be detected by any suitable means such asspectrophotometry.

The immunoassay may be, without limitation, in a heterogenous or in ahomogeneous format, and of a standard or competitive type. In aheterogeneous format, the targetable construct or complex is typicallybound to a solid support to facilitate separation of the sampletherefrom after incubation. The solid support containing the targetableconstruct or complex is typically washed after separating it from thetest sample, and prior to detection of bound antigens. In a homogeneousformat, the test sample is incubated with the combination of targetableconstructs or complexes in solution. For example, it may be underconditions that will precipitate any targetable complex/antigenassemblages that are formed. Both standard and competitive formats forthese assays are known in the art.

In a standard format, the amount of antigen bond to the immobilizedtargetable construct or complex is directly monitored. This may beaccomplished, for example, by detecting labeled anti-xenogenic (e.g.,anti-human) antibodies that recognize an epitope on the bsAbs ortargetable construct. In a competitive format, the amount of antigens ina sample is deduced by monitoring the competitive effect on the bindingof a known amount of labeled antigen (or other competing ligand) addedto the sample before or during the assay.

Targetable constructs or complexes may be immobilized to the innersurface of microtiter wells and the test sample and prelabeled targetepitopes added to the wells. After a select period, the wells are washedand the color developed on the floor of the wells from theantibody-antigen reaction examined. By use of an automatic reader, theresults of numerous tests can be determined in a few minutes.

II.D.2. Immunoassay Kits

The targetable complexes may be packaged in the form of a kit for use inimmunoassays. The kit contain in separate containers the separatecombination of targetable constructs, targetable complexes and bsAbs(either already bound to a solid matrix or separate with reagents forbinding them to the matrix), control antibody formulations (positiveand/or negative), labeled antibody when the assay format requires sameand signal generating reagents (e.g., enzyme substrate) if the labeldoes not generate a signal directly. Instructions (in any of a number offormats, e.g., written, tape, VCR, CD-ROM, etc.) for carrying out theassay may be included in the kit.

Test kits according to the invention for comprising a solid support thatis an immunoassay contain, for example, a suitable container, coatedwith a targetable construct or targetable complex of the invention,optionally freeze-dried or concentrated solutions of a targeted epitopeand/or a labelled derivative thereof, standard solutions of thisprotein, buffer solutions and, optionally, polypeptides and detergentsfor preventing non-specific adsorption and aggregate formation,pipettes, reaction vessels, calibration curves, instruction manuals andthe like.

II.D.3. Dipsticks

The targetable constructs and complexes may be used in a “dipstick” orsheet which is capable of being inserted into and withdrawn from asample. The dipstick is dipped into a well mixed urine sample, and aftera time period of thirty seconds to two minutes, the various reagentbands are visually or optically examined for color changes. The bandscan be visually compared to a preprinted color chart in order todetermine the amount of each of the constituents or parameters beingmeasured.

In a typical dipstick based analytical assay, a ligand, whichspecifically binds to the analyte of interest, is bound to a solidsupport on the dipstick. The dipstick is contacted with a sample inwhich the presence of the analyte of interest is to be determined.Frequently, steps are employed to aid in the removal of non-specificallybound material from the dipstick. Finally, the dipstick is processed todetermine the presence of the analyte. The dipstick generally comprisesa solid material, which is planar or columnar in geometry.

II.E. Ex vivo Therapeutic Modalities

The compositions and devices of the invention, and methods of usethereof, may be used in ex vivo modalities. An “ex vivo modality” is onein which a biological sample, such as a body fluid, is temporarilyremoved from an animal, altered through in vitro manipulation designedto remove or inactivate one or more undesirable substances, and thenreturned to the body. One way in which undesirable substances may beremoved from the sample is by contacting the sample with an agent thatbinds the undesirable substance. In an ex vivo modality of theinvention, a sample that has been temporarily removed from a patient iscontacted with a solid support comprising a targetable complex of theinvention. In this embodiment, the undesirable substance is or comprisesa target epitope that is recognized by the targetable complex of thesolid support. The undesirable substance is, or is part of, e.g., atoxin, a hyperproliferative cell, an infected cell or a pathogen. Forexample, for the treatment of viremia, a virus that comprises a targetepitope is cleared from blood by contacting the blood with a solidsupport comprising a targetable complex of the invention. The targetablecomplex recognizes and binds the virus, which is retained in thedialysis system but not in the blood that is returned to the patient.

An exemplary ex vivo modality of the invention is a hemodialysis system,which comprises a dialysis machine. A “dialysis machine” is a device inwhich a fluid such as blood of an animal is temporarily removedtherefrom and processed through one or more physical, chemical,biochemical or other types of processes designed to remove or inactivateundesirable substances. Bodily waste products, toxins, venoms,overexpressed or overactive endogenous agents, molecules derived fromany of the preceding, and pathogens comprising any of the preceding, arenon-limiting examples of undesirable substances. In an exemplary mode, ahuman is treated by a dialysis machine that augments or substitutes forthe natural kidney functions of a human body. Blood is removed from thebody, passed through the dialysis machine, which separates the wastesfrom the blood extracorporeally. The separated wastes are discharged anddisposed of, whereas the treated blood is returned to the body.

The transfer of blood between the patient and the dialyzer occurs withina blood tubing set that is usually disposable. The blood tubing set andthe dialyzer represent a closed extracorporeal path through which apatient's blood travels. The blood tubing set includes an arterial lineconnected to an arterial reservoir for drawing blood from a patient, avenous line connected to a venous reservoir for returning blood to thepatient, and a number of other lines for connecting a pump and thedialyzer between the arterial and venous reservoirs. Before the bloodtubing set and the dialyzer can be used in a dialysis treatment, bothmust be primed with a sterile saline solution to remove air from theextracorporeal circuit. Once primed, the saline solution is recirculatedthrough the blood tubing set and the dialyzer to produce a stabilizedflow and remove additional trapped air from within the extracorporealcircuit. The priming and recirculating process also serves to clean thedialyzer and flush the dialyzer membrane of any debris or chemicalsremaining from a prior use.

A commonly used method of creating blood access for hemodialysis is bymeans of an arteriovenous fistula. For each dialysis session, thefistula must be punctured with large bore needles to deliver blood into,and return blood from, the artificial kidney (dialyzer). Even with theuse of anesthetics, the punctures with these large bore needles arepainful. Patients undergoing dialysis thus benefit if the punctures canbe done as infrequently as possible. Moreover, frequent punctures may bedetrimental to the longevity of the fistula.

Existing hemodialysis systems consist fundamentally of two halves; onecomprising the extracorporeal blood circuit (the blood flow path) andthe other comprising the dialysate circuit or flow path. Typically, theentire blood circuit is disposable and comprises: (1) an arterial andvenous fistula needle, (2) an arterial (inflow) and venous (outflow)blood line, (3) a hemodialyzer, (4) one or more physiologic primingsolutions (e.g., saline), and (5) one or more anticoagulants (e.g.,heparin or citrate).

The arterial fistula needle accesses blood from the patient's fistulaand is connected to the arterial blood tubing set, which conveys bloodto the dialyzer. The arterial line comprises a pumping segment withinterfaces to a blood pump (which may be, e.g., a rotary or peristalticpump) on the dialysis machine, pressure or flow monitoring chambersincluding tubing which interfaces to pressure or flow transducers on themachine to monitor the pressure and flow pre-pump and/or post pump,inlet ports for saline and anticoagulant, and one or more injectionsites for, e.g., drawing blood or injecting drugs.

The hemodialyzer typically comprises a case which encloses a bundle ofhollow fiber semi-permeable membranes, which are usually made fromcellulose or synthetic polymers. The blood is circulated on one side ofa semipermeable membrane, and the dialysis solution is circulated on theother side, so that the two never come into direct contact. Wasteproducts (uremic toxins) diffuse out of the blood, across thesemipermeable membranes, and into the dialysis solution owing to theconcentration gradient. Excess water in the patent's blood enters thedialysate as a result of a pressure gradient.

The venous blood line and venous fistula needle carry the newly dialyzedblood away from the dialyzer and back into the patient's circulatorysystem via a puncture site slightly closer to the heart than thearterial needle site. The venous set is comprised of a pressuremonitoring chamber with tubing leading to another pressure transducer inthe machine, injection sites, and a segment of tubing which interfacesto an air detection assembly in the machine in order to prevent airemboli during treatment.

A dialysis machine has several systems and components. In anextracorporeal flow path, which conducts blood from the patient to thedialyzer and then back to the patient, at least one arterial blood pumpand sometimes a venous blood pump that move the blood and assist inperforming certain types of dialysis treatment such as ultrafiltration.A hydraulics flow path, which conducts the dialysate through thedialyzer, includes numerous components to monitor and control theconditions in that flow path. Flow and pressure meters may be included,typically at the inlet and outlet of the dialyzer. A first dialysatepump moves dialysate into the dialyzer, and a second dialysate pumpremoves the dialysate from the dialyzer. A heater may be included toheat the dialysate to body temperature to avoid undesirable heattransfer to or from the patient, and/or to heat a disinfecting solutionto temperatures adequate to kill microorganisms. Other components may beincluded in dialysis machines designed for specific applications. Forexample, in ultrafiltration dialysis treatments, an ultrafiltration pumpis used to control the delivery of desirable components to the blood.

Proportioning pumps for one or more dialysis solutions may be included.Dialysis solution is typically prepared continuously on-line inpresent-day machines by combining water which has first been purified bya separate water treatment system, and liquid concentrates ofelectrolytes. Dialysate concentrates have evolved, from a singleformulation which contained acetate as the physiologic buffering agentfor the correction of circulatory acidosis, to two container systemswhere bicarbonate replaces acetate as the buffering agent, and must bekept separate due to its chemical incompatibility with calcium andmagnesium. Two proportioning pumps are therefore required, the first tomix the bicarbonate concentrate with water and the second to proportionthis mixture with the concentrated electrolytes to achieve the final,physiologically compatible dialysis solution.

The dialysis machine continuously monitors the pressure at the bloodinlet and outlet sides of the dialyzer (by way of the pressuretransducers connected to the blood sets) as well as in the dialysatecircuit. Via microprocessors, the system calculates the transmembranepressure (TMP) which determines the amount of water transmission throughthe membranes. Dialysis machines may also comprise a device formeasuring the amount of dialysis solution entering and dialysate leavingthe dialyzer, which allows the calculation of net water removal from thepatient. By electronically comparing the amount of water entering orleaving the blood with the TMP, the system is able to control activelythe water removed from the patient to a desired target previouslyprogrammed into the system. When low-water-transmission cellulosicmembranes are employed, negative pressure is generated on the dialysateside of the membrane by the machine in order to accomplish sufficientwater removal. Because suction may be applied to the dialysate as ittransits the dialyzer, it is first be placed under a greater vacuum in adegassing chamber so that air bubbles are not generated within thedialyzer that would cause errors in the calculation of ultrafiltrationby the flow sensors and also reduce the efficiency of the dialyzer. Incontrast, when high-water-transmission, synthetic membranes are used, itis frequently necessary to apply positive pressure on the dialysate sideto control the rate of ultrafiltration.

Another non-limiting example of ex vivo therapeutic applications of theinvention is the use of the compositions of the invention in devices forthe intraoperative and post-surgical salvaging of blood. In thisembodiment, a patient's own blood lost during intraoperative and/orpost-surgical procedures, is washed and filtered and then reinfused intothe patient. For a non-limiting examplary device of this type, see U.S.Pat. No. 5,876,611.

III. Molecular Scaffolds and Targetable Constructs

II.A. Structure of Molecular Scaffolds and Targetable Constructs

The targetable construct(s) present in a targetable complex comprises amolecular scaffold which comprises or bears at least two pairs ofcarrier epitopes recognized by the arm of an antibody or antibodyfragment in the complex.

The targetable construct can be of diverse structure, but is selectednot only to elicit sufficient immune responses, but also for rapid invivo clearance when used within the bsAb targeting method. Exemplarytargetable constructs for use in the present application are describedin U.S. application Ser. No. 09/337,756 filed Jun. 22, 1999, and in U.S.application Ser. No. 09/823,746, filed Apr. 3, 2001, the entire contentsof which are incorporated herein by reference.

Hydrophobic agents are best at eliciting strong immune responses,whereas hydrophilic agents are preferred for rapid in vivo clearance,thus, a balance between hydrophobic and hydrophilic needs to beestablished. This may be accomplished in a preferred approach, in part,by relying on the use of hydrophilic chelating agents to offset theinherent hydrophobicity of many organic moieties. Also, sub-units of thetargetable construct may be chosen which have opposite solutionproperties, for example, peptides, which contain amino acids, some ofwhich are hydrophobic and some of which are hydrophilic. Aside frompeptides, carbohydrates may be used. Additionally, the targetableconstruct can comprise PEG (poly[ethylene] glycol) derivatives toincrease its circulation time in a patient.

Peptides having as few as one amine residue may be used, preferably twoto ten amino acid residues, if also coupled to other moieties such aschelating agents. Examples include modified amino acids, such asbis-DTPA-lysine, and bis-DTPA-diamine.

These agents can be linked covalently to molecules which are to betargeted. The hapten moiety of the carrier portion should be a lowmolecular weight conjugate, preferably having a molecular weight of100,000 daltons or less, and advantageously less than about 20,000daltons, 10,000 daltons or 5,000 daltons, including the metal ions inthe chelates. For instance, the known peptidedi-indium-DTPA-Tyr-Lys(DTPA)-OH has been used to generate antibodiesagainst the indium-DTPA portion of the molecule. However, by use of thenon-indium-containing molecule, and appropriate screening steps, new Absagainst the tyrosyl-lysine dipeptide can be made. More usually, theantigenic peptide will have four or more residues, such as the peptideAc-Phe-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂. Again, the non-metal-containingpeptide is used as an immunogen, with resultant Abs screened forreactivity against the Phe-Lys-Tyr-Lys backbone. Another non-limitingexample of an antigenic peptide having four or more residues is thepeptide DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂, wherein DOTA is1,4,7,10-tetraazacyclododecanetetraacetic acid and HSG is the histaminesuccinyl glycyl group of the formula:

The non-metal-containing peptide may be used as an immunogen, withresultant Abs screened for reactivity against the Phe-Lys-Tyr-Lysbackbone.

In one embodiment, unnatural amino acids, e.g., D-amino acids, areincorporated into the backbone structure to ensure that, when used withthe final bsAb/linker system, the scFv component which recognizes thelinker moiety is completely specific. The invention further contemplatesother backbone structures such as those constructed from non-naturalamino acids, peptoids, peptidomimetics, aptamers, peptide nucleic acids(PNAs), and the like.

According to one embodiment of the invention, the targetable constructcan encompass a carbohydrate. Suitable such carbohydrates includecarbohydrate chains of two to six sugar units long. The targetableconstruct also can comprise a polymeric carbohydrate, such as dextran.

In another embodiment of the invention, the haptens of the targetableconstruct comprise a known immunogenic recognition moiety, for example,a known hapten. Using a known hapten, for example, fluoresceinisothiocyanate (FITC), higher specificity of the targetable constructfor the antibody is exhibited. This occurs because antibodies raised tothe hapten are known and can be incorporated into the inventiveantibody. Thus, binding of the targetable construct with the attachedchelator or metal-chelate complex would be highly specific for theinventive antibody or antibody fragment. Another example of a hapten tobe substituted onto the targetable construct includes vitamin B12. Theuse of vitamin B12 is advantageous since anti-B12 Mabs are known and nofree serum B12 exists, therefore, great specificity for the antibody maybe exhibited. The chelator or its chelate with a metal cation also canfunction as the hapten to which an antibody is raised. Another exampleof a hapten to be conjugated to a targetable construct includes biotin.

III.B. Preparation of Molecular Scaffolds and Targetable Constructs

Peptides, including but not limited to, peptides to be used as molecularscaffolds or immunogens are synthesized conveniently on an automatedpeptide synthesizer using a solid-phase support and standard techniquesof repetitive orthogonal deprotection and coupling.

Free amino groups in the peptide that are to be used later for chelateconjugation are advantageously blocked with standard protecting groupssuch as an Aloc group. Such protecting groups will be known to theskilled artisan. See Greene and Wuts, Protective Groups in OrganicSynthesis, John Wiley and Sons, New York (1999) and Kates S A, AlbericioF. Solid-Phase Synthesis: A Practical Guide. Marcel Dekker, New York(2000). Methods of synthesizing amino acid-based polymers andglycosylated peptides are described in, respectively, Sanda and Endo,Syntheses and Functions of Polymers Based on Amino Acids, Macromol.Chem. Phys. 200:2651-2661, 1999; and Sears and Wong, Toward automatedsynthesis of oligosaccharides and glycoproteins, Science 291:2344-2350,2001.

III.C. Chemical Conjugation

Molecular scaffolds may be prepared as a single molecule, or may begenerated by first preparing subunits that are then covalently attachedto each other. The term “conjugation” is used to indicate the covalentattachment of two or more molecules.

Peptides are conjugated (i.e., linked, or covalently attached), to oneanother using various methods. By way of non-limiting example, aminoacid residues present in the natural sequence of a first peptide can bedirectly covalently linked to amino acid residues in the natural aminoacid sequence of a second peptide as in, e.g., a disulfide bridge; or across-linking reagent (also known as “cross-linker”), typically abifunctional (two-armed) chemical linker that forms covalent linkagesbetween two or more peptides, can be used to covalently link peptides toeach other. Such bifunctional linkers can be homobifunctional (whereinboth “arms” of the linker are the same chemical moiety) orheterobifunctional (wherein each of the two “arms” is a differentchemical moiety than the other).

Hermanson (Bioconjugate Techniques, Academic Press, 1996), hereinincorporated by reference, summarizes many of the chemical methods usedto link proteins and other molecules together using various reactivefunctional groups present on various cross-linking or derivatizingreagents. Cross-linking agents are based on reactive functional groupsthat modify and couple to amino acid side chains of proteins andpeptides, as well as to other macromolecules. Cross-linking reagentsincorporate two or more functional reactive groups. The functionalreactive groups in a cross-linking reagent may be the same or different.Many different cross-linkers are available to cross-link variousproteins, peptides, and macromolecules. Table I lists some of thecross-linkers that are easily available through commercial sourcesaccording to their class of chemical reactivity. Table 2 lists some ofthe properties of chemical cross-linkers and the types of functionalgroups with which they react. TABLE 1 CLASSES OF CHEMICAL REACTIVITY OFCROSS-LINKERS AND EXAMPLES OF CROSS-LINKERS Chemical reactivityAbbreviation Compound homobifunctional DMA Dimethyl adipimidate.2 HClimidoesters DMP Dimethyl pimelimidate.2 HCl DMS Dimethyl suberimidate.2HCl DTBP Dimethyl 3,3′-dithiobispropionimidate.2HCl homobifunctional N-DSG Disuccinimidyl glutarate hydroxysuccinimide esters (NHS-esters) DMSCDimethyl succinimidate.2 HCl DSS Disuccinimidyl suberate BS DSPDithiobis(succinimidylpropionate) DTSSPDithiobis(sulfosuccinimidylpropionate) DTME Dithio-bis-maleimidoethaneEGS Ethylene glycolbis(succinimidylsuccinate) Sulfo-EGS Ethyleneglycolbis(sulfosuccinimidylsuccinate) DST Disuccinimidyl tartrateSulfo-DST Disulfosuccinimidyl tartrate BSOCOES Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone Sulfo- Bis[2- BSCOCOES(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone heterobifunctional BS3BIS-(sulfosuccinimidyl) suberate NHS-esters DMM dimethyl malonimidate.2HCl EMCS N-[ε-maleimidocaproyloxy]succinimide ester Sulfo-EMCSN-[ε-maleimidocaproyloxy]sulfosuccinimide ester SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate LC-SMCC succiminidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6- amido-caproate) Sulfo-MBSm-maleimidobenzoyl-N- hydoxysulfosuccinimide ester Sulfo-SMCCsulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate MBSm-maleimidobenzoyl-N-hydoxysuccinimide ester SMPB succinimidyl4-[P-Maleimidophenyl] butyrate Sulfo-SMPB sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate BMH bismaleimidohexane GMBSN-[γ-Maleimidobutyryloxy] succinimide ester Sulfo-GMBSN-[γ-Maleimidobutyryloxy] sulfosuccinimide ester heterobifunctional SIABN-succinimidyl(4-iodoacetyl)aminobenzoate haloacetyl NHS-estersSulfo-SIAB Sulfo-SIAB sulfosuccinimidyl(4- iodoacetyl)aminobenzoatehomobifunctional DPDPB 1,4-Di-[3′-(2′- pyridyldithiolspyridyldithio)propionamido]butane heterobifunctional SMPT4-succinimidyloxycarbonyl-methyl-(2- pyridyldithiolspyridyldithio)-toluene Sulfo-LC- sulfosuccinimidyl6-[a-methyl-a-(2-pyridyl- SMPT dithio)toluamido]hexanoate SPDPN-succinimidyl 3-(2-pyridyldithio)propionate LC-SPDP N-succinimidyl6-[3′-(2- pyridyldithio)propionamido]hexanoate Sulfo-LC-sulfosuccinimidyl 6-[3′-(2-pyridyldithio)- SPDP propionamido] hexanoatecarboxyl reactive PDPH 3-(2-Pyridyldithio) propionyl hydrazide carbonylreactive EDC 1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide M2C2H4-(N-Maleimidomethyl)cyclohexane-1- carboxyl hydrazide DCCN,N-dicyclohexylcarbodimide MPBH 4-(4-N-Maleimidophenyl)butyric acidhydrazide hydrochloride Photoreactive ABH Azidobenzoyl hydrazide ANB-NOSN-5-azido-2-nitrobenzoyloxysuccinimide APDPN-[4-(p-azidosalicylamido)butyl]-3′(2′- pyridyldithio)propionamide APGp-Azidophenylglyoxal monhydrate ASBA 4-(p-Azidosalicylamido)butylamineASIB 1-(p-Azidosalicylamido)-4- (iodoaceamido)butane BASEDBis-[B-4-azidosalicylamido)ethyl]disulfide HSABN-Hydroxysuccinimidyl-4-azidobenzoate Sulfo-HSABN-Hydroxysulfo-succinimdyl-4-azidobenzoate NHS-ASAN-Hydroxysuccinimidyl-4-azidosalicylic acid Sulfo-NHS-N-Hydroxysulfo-succinimidly-4-azidosalicylic ASA acid Sulfo-NHS-Sulfosuccinimidly-[4-azidosalicylamido)- LC-ASA hexanoate PNP-DTPp-Nitropheyno-2-diazo-3,3,3- trifluoropropionate DTP2-Diazo-3,3,3-trifluoropropionylchloride SADPN-succinimidyl-(4-azidopheynyl 1,3′ dithiopropionate Sulfo-SADPSulfosuccinimidyl-(4- azidophynyldithio)propionate SAEDSulfosuccinimidyl 2(7-azido-4- methylcoumarin-3-acetamide)ethyl-1,3-dithiopropionate Sulfo- Sulfosuccinimidyl 7-azido-4-methycoumarin- SAMCA3-acetate SAND Sulfosuccinimidyl 2-(m-azido-o-nitrobenzamdio)-ethyl-1,3-dithiopropionate SANPHN-succinimidyl-6-(4′-azido-2′- nitrophenylamino)hexanoate Sulfo-Sulfosuccinimidyl 6-(4′-azido-2′- SANPH nitrophenylamino)hexanoate SASDSulfosuccinimidyl 2-(p- azdiosalicylamido)ethyl-1,3′-dithiopropionateSulfo-SAPB Sulfosuccinimidyl 4-(p-azidophenyl)-butyrateHeterobifunctional SDBP N-Hydroxysuccinimidyl 2,3- amine reactivedibromopropionate Bifunctional aryl DFDNB1,5-Difluoro-2.4-dinitrobenzene halide heterobifunctional mal-sac-maleimido-6-aminocaproyl-ester of nitrophenylsulfonic HNSA1-hydroxy-2-nitrobenzene-4-sulfonic acid acid ester

TABLE 2 CHEMICAL CROSS-LINKERS AND SOME OF THEIR PROPERTIES PierceSpacer Arm Product Length Cleavable Water Membrane Acronym Number(angstroms) Links By Soluble Permeable Sulfo-LC-SMPT 21568 20.0 AminesTo Thiols Yes No Sulfhydryls SMPT 21558 20.0 Amines To Thiols Yes NoSulfhydryls Sulfo-KMUS 21111 19.0 Amines To non Yes No SulfhydrylsLC-SMCC 22362 16.1 Amines To non Yes No Sulfhydryls KMUA 22211 15.7Amines To non Yes No Sulfhydryls LC-SPDP 21651 15.6 Amines To non No ndSulfhydryls Sulfo-LC-SPDP 21650 15.6 Amines To Thiols Yes No SulfhydrylsSMPB 22416 14.5 Amines To non No Yes Sulfhydryls Sulfo-SMPB 22317 14.5Amines To non Yes No Sulfhydryls SMPH 22363 14.3 Amines To non No ndSulfhydryls SMCC 22360 11.6 Amines to non No Yes Sulfhydryls Sulfo-SMCC22322 11.6 Amines to non Yes No Sulfhydryls SIAB 22329 10.6 Amines tonon No Yes Sulfhydryls Sulfo-SIAB 22327 10.6 Amines To non Yes NoSulfhydryls Sulfo-GMBS 22324 10.2 Amines To non Yes No Sulfhydryls GMBS22309 10.2 Amines To non No Yes Sulfhydryls MBS 22311 9.9 Amines To nonNo Yes Sulfhydryls Sulfo-MBS 22312 9.9 Amines To non Yes No SulfhydrylsSulfo-EMCS 22307 9.4 Amines To non Yes No Sulfhydryls EMCA 22306 9.4Amines To non Yes No Sulfhydryls EMCS 22308 9.4 Amines To non No YesSulfhydryls SVSB 22358 8.3 Amines To non No Yes Sulfhydryls BMPS 222986.9 Amines To non No nd Sulfhydryls SPDP 21857 6.8 Amines To Thiols NoYes Sulfhydryls SBAP 22339 6.2 Amines To non No Yes Sulfhydryls BMPA22296 5.9 Amines To non Yes No Sulfhydryls AMAS 22295 4.4 Amines To nonNo nd Sulfhydryls SATP 26100 4.1 Amines To non No Yes Sulfhydryls SIA22349 1.5 Amines To non No nd Sulfhydryls Sulfo-LC-SMPT 21568 20.0Sulfhydryls Thiols Yes No to Amines SMPT 21558 20.0 Sulfhydryls ThiolsNo Yes to Amines AEDP 22101 9.5 Carboxyls Thiols Yes No to Amines EDC22980 0.0 Carboxyls non Yes No to Amines

Bifunctional cross-linking reagents may be classified according to theirfunctional groups, chemical specificity, length of the cross bridge thatthey establish, the presence of similar functional groups or dissimilarfunctional groups, chemical or photochemical reactivity, ability to becleaved internally by reduction or other means, and the ability of thereagent to be further modified by radiolabelling (i.e. radioiodination)or addition of detectable tags or labels. The selective groups on thecross-linking reagent can be present in a homobifunctional arrangementin which the selective groups are identical, or can be present in aheterobifunctional arrangement in which the selective groups aredissimilar.

The chemical modification may be done using cross-linking reagentscontaining selective groups that react with primary amines, sulfhydryl(thiol) groups, carbonyl, carboxyl groups, hydroxyl, or carbohydratesand other groups placed on a protein or peptide, especially byposttranslational modifications within the cell. The selective groupsinclude, but are not limited to, imidoester, N-hydroxysuccinimide esteror sulfosuccinimidyl ester, ester of1-hydroxy-2-nitrobenzene-4-sulfonic, maleimide, pyridyl disulfide,carbodiimide, and haloacetyl groups.

Sulfhydryl reactive functional groups include maleimides, alkyl and arylhalides, haloacyls, haloacetyls and pyridyl disulfides. Maleimides,alkyl and aryl halides, haloacetyls and haloacyls react with thiols toform stable thioether bonds that are not reduced by reagents such as2-mercaptoethanol and dithiothreitol. Pyridyl disulfides form mixeddisulfides with thiol groups, mixed disulfides may be used as anintermediate for cross-linking two or more macromolecules. Cross-linkersthat first react with a carboxyl group to form an activated intermediateand then reacts with an amino group, such as an amino group of lysine oran amino group of an amino terminal amino acid, may be used.

A spacer arm or “cross-bridge” region, consisting of a spacer group or afunctional group, such as a disulfide bond or hindered disulfide bond,connects the two selective or functional groups. The length of thespacer arm may be varied. The distance between the functional groupsestablishes the length of the spacer arm. Longer spacer arms may berequired to diminish or eliminate steric hindrance between two moleculesthat are cross-linked together. Intermolecular cross-linking is moreefficient with longer spacer arms. Short spacer arms favorintramolecular cross-linking, which is to be avoided in the presentinvention.

Spacer arms may have reactive bonds within them that enable furthermodifications. For example, internal cleavable bonds may be placedwithin the spacer, such as disulfides or hindered disulfides, one ormore ester bonds, or vicinal hydroxyl groups. Cleavage of internaldisulfide bonds may be achieved using reduction with thiol containingreagents such as 2-mercaptoethanol and dithiothreitol. One or moremetabolizable bonds may be inserted internally in the cross-linkingreagent to provide the ability for the coupled entities to separateafter the bond(s) is broken after the conjugate is transported into thecell and into the body.

Homobifunctional cross-linkers contain at least two identical functionalgroups. Heterobifunctional cross-linkers contain two or more functionalreactive groups that react with different specificity. Becauseheterobifunctional cross-linkers contain different reactive groups, eachend can be individually directed towards different functional groups onproteins, peptides, and macromolecules. This feature results in linking,for example, amino groups on one molecular entity to carboxyl groups onanother entity, or amino groups on one entity to sulfhydryl groups onanother entity.

Functional groups include reactive portions on proteins, peptides, andmacromolecules that are capable of undergoing chemical reaction.Functional groups include amino and carboxyl groups, hydroxyl groups,phenolate hydroxyl groups, carbonyl groups, guanidinyl groups, andcarbon-carbon double bonds. In addition, photoactive reagents thatbecome reactive when exposed to light may be used. For example,arylazides may be activated to form activated intermediates, such as anaryl nitrene or a dehydroazepine intermediate, that non-selectivelyinserts into carbon-hydrogen bonds (i.e. by aryl nitrenes) or reactswith amines (dehydroazepines). Other examples include fluorinated arylazides, benzophenones, certain diazo compounds, and diazrinederivatives.

If the desired sulfhydryl groups are not present on the protein, peptideor macromolecule, a sulfhydryl may be introduced by chemicalmodification. As a nonlimiting example, the sFv or a therapeuticmacromolecule can be modified so as to introduce a thiol by chemicalmodification. A cysteine amino acid can be placed in a peptide duringpeptide synthesis. Sulfhydryl groups can be added by chemicalmodification using 2-iminothiolane (IT), also known as Traut's reagent.

Sulfhydryl groups can also be added by using a modification reagent thatcontains a disulfide bond in addition to a group that selectively reactswith primary amines. For example, the heterobifunctional cross-linkersulfosuccinimidyl 6-[3′-(2-pyridyldithio)-propionamido] hexanoate(sulfo-LC-SPDP, Pierce Chemical Co.) will thiolate peptides when usedaccording to the manufacturer's directions. Other, non-soluble, formssuch as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP, PierceChemical Co.) or N-succinimidyl6-[3′-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP, Pierce ChemicalCo.) can be used in these reactions by dissolving in a suitable organicsolvent to a concentration of 20 mM, and adding 25-1 to 1 ml of 10 mg/mlpeptide. Reducing the SPDP-derivatized peptide under mild conditionswill release pyridine-2-thione, leaving an aliphatic thiol. An exampleof a mild reducing condition is to add {fraction (1/100)}th volume of 1Mdithiothreitol (DTT) to the above SPDP-derivatized target peptide andincubating for 30 minutes at room temperature, or incubate theSPDP-derivatized target peptide with 50 mM 2-meraptoethylamine inPBS-EDTA for 90 minutes at 37° C. The excess SPDP, LC-SPDP orsulfo-LC-SPDP, and the pyridine-2-thione can then be removed by HPLCpurification.

These modification reagents may also contain groups near the added thiolsuch that they form a hindered disulfide when oxidized. These reagents,such as 4-succinimidyloxycarbonyl-methyl-(2-pyridyldithio)-toluene(SMPT), may result in a conjugate that exhibits increased stability invivo (Thorpe et al. Cancer Res. 47:5924-5931, 1987). Other cross-linkingreagents can be used for protein thiolation and are known to those wellversed in the art. Many of these reagents are described in the PierceChemical Co. catalog, or by Ji, Meth. Enzymol. 91:580-609, 1983; andHermanson, Bioconjugate Techniques, Academic Press, Inc., San Diego,1-785, 1996.

Most commonly, carrier molecule scaffold portions will have eithersulfhydryl or primary amines as the targets of the cross-linkingreagents, and both sulfhydryl and primary amines can either existnaturally or be the result of chemical modification as described above.When both carrier molecular scaffold portions have a reduced sulfhydryl,a homobifunctional cross-linker that contains maleimide, pyridyldisulfide, or haloacetyl groups can be used for cross-linking. Examplesof such cross-linking reagents include, but are not limited to,bismaleimidohexane (BMH) or1,4-Di-[3′-(2′-pyridyldithio)propionamido]butane (DPDPB). Alternatively,a heterobifunctional cross-linker that contains a combination ofmaleimide, pyridyl disulfide, or haloacetyl groups can be used forcross-linking. Less preferably, the cross-linking reagent can containthiophthalimide derivatives or disulfide dioxide derivatives. Also,extrinsic sulfhydryl groups can be introduced into the carrier molecularscaffold portions and oxidized to cross-link by disulfide formation.

When primary amines are selected as the target both on sFv andtherapeutic macromolecule, then a homobifunctional cross-linker thatcontains succinimide ester, imidoester, acylazide, or isocyanate groupscan be used for cross-linking. Examples of such cross-linking reagentsinclude, but are not limited to, Disuccinimidyl glutarate (DSG),Dithiobis(succinimidylpropionate) (DSP),Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES),Bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSCOCOES),Disuccinimidyl suberate (DSS), Bis-(Sulfosuccinimidyl) Suberate (BS3),Disuccinimidyl tartrate (DST), Disulfosuccinimidyl tartrate (sulfo-DST),Dithio-bis-maleimidoethane (DTME), Ethyleneglycolbis(succinimidylsuccinate) (EGS),Dithiobis(sulfosuccinimidylpropionate) (DTSSP), Ethyleneglycolbis(sulfosuccinimidylsuccinate) (sulfo-EGS), Dimethylmalonimidate•2 HCl (DMM), Dimethyl succinimidate•2 HCl (DMSC), Dimethyladipimidate•2 HCl (DMA), Dimethyl pimelimidate•2 HCl (DMP), Dimethylsuberimidate•2 HCl (DMS), and Dimethyl 3,3′-dithiobispropionimidate•2HCl(DTBP). Heterobifunctional cross-linkers that contains a combination ofimidoester or succinimide ester groups can also be used forcross-linking.

Heterobifunctional cross-linking reagents that combine selective groupsagainst different targets are generally preferred because these allowreactions to be performed selectively and sequentially, minimizingself-association or polymerization. Also, heterobifunctional reagentsallow selection of chemistry appropriate for the individual molecules tobe joined. Examples of such cross-linking reagents include, but are notlimited to, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl 6-[3′-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP),sulfosuccinimidyl 6-[3′-(2-pyridyldithio)-propionamido] hexanoate(sulfo-LC-SPDP), m-maleimidobenzoyl-N-hydoxysuccinimide ester (MBS),m-maleimidobenzoyl-N-hydoxysulfosuccinimide ester (sulfo-MBS),succinimidyl 4-[P-maleimidophenyl] butyrate (SMPB), sulfosuccinimidyl4-[p-maleimidophenyl] butyrate (sulfo-SMPB), N-[Maleimidobutyryloxy]succinimide ester (GMBS), N-[maleimidobutyryloxy] sulfosuccinimide ester(sulfo-GMBS), N-[maleimidocaproyloxy] succinimide ester (EMCS),N-[maleimidocaproyloxy] sulfosuccinimide ester (sulfo-EMCS),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB),sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB), succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC),succiminidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amido-caproate)(LC-SMCC), 4-succinimidyloxycarbonyl-methyl-(2-pyridyldithio) toluene(SMPT), and sulfo-LC-SMPT.

IV. Chelate Moieties

The presence of hydrophilic chelate moieties on the targetable constructhelps to ensure rapid iii vivo clearance. In addition to hydrophilicity,chelates are chosen for their metal-binding properties, and are changedat will since, at least for those targetable constructs whose bsAbepitope is part of the peptide or is a non-chelated hapten, recognitionof the metal-chelate complex is no longer an issue.

The nature of the invention is such that several chelate moities may beused in a targetable construct or complex. For example, if two types ofbsAbs are to be used in a complex, each of which has a different bindingspecificity for a carrier epitope (i.e., each of which binds a differenttype of chelate moiety), then the targetable construct comprises bothtypes of chelate moieties. Those skilled in the art will be able tochoose appropriate chelate moieties depending on the nature andstructure of the targetable construct and bsAbs to be used, and theintended application of the targetable constructs and complexes. If needbe, scFvs having specificities for novel carrier epitopes can begenerated and incorporated into a bsAb.

Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, used with ⁴⁷Sc, ⁵²Fe, ⁵⁵Co, ⁶⁷Ga,⁶⁸Ga, ¹¹¹In, ⁸⁹Zr, ⁹⁰Y, ¹⁶¹Tb, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, and ²²⁵Ac forradio-imaging and RAIT. The same chelators, when complexed withnon-radioactive metals, such as manganese, iron and gadolinium can beused for MRI, when used along with the bsAbs of the invention.Macrocyclic chelators such as NOTA, DOTA, and TETA are of use with avariety of metals and radiometals, most particularly with radionuclidesof gallium, ytrrium and copper, respectively.

DTPA and DOTA-type chelators, where the ligand includes hard basechelating functions such as carboxylate or amine groups, are mosteffective for chelating hard acid cations, especially Group IIa andGroup IIIa metal cations. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelators such as macrocyclic polyethers, which are ofinterest for stably binding nuclides such as ²²³Ra for RAIT areencompassed by the invention. Porphyrin chelators may be used withnumerous radiometals, and are also useful as certain non-radioactivemetal complexes for bsAb-directed immuno-phototherapy. More than onetype of chelator may be conjugated to a carrier to bind multiple metalions, e.g., non-radioactive ions and/or radionuclides. One example is abis-¹¹¹In-DTPA conjugate that also bears a DOTA-⁹⁰Y chelate.Particularly useful therapeutic radionuclides include, but are notlimited to ³²P, ³³P, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, Ga, ⁹⁰Y, ¹¹¹Ag, ¹¹¹In, ¹²⁵I,¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶R, ¹⁸⁸Re, ¹⁸⁹Re,²¹²Pb, ²¹³Bi ²¹¹At, ²²³Ra and ²²⁵Ac.

Examplary radioactive metal chelate complexes have been described thatuse radionuclides such as cobalt-57 (Goodwin et al., U.S. Pat. No.4,863,713), ¹¹¹In (Barbet et al., U.S. Pat. No. 5,256,395 and U.S. Pat.No. 5,274,076; Goodwin et al., J. Nucl. Med., 33:1366-1372, 1992;Kranenborg et al., Cancer Res. (suppl.), 55:5864s-5867s, 1995, andCancer (suppl.) 80:2390-2397, 1997) and ⁶⁸Ga (Boden et al., BioconjugateChem. 6:373-379, 1995; and Schuhmacher et al., Cancer Res. 55:115-123,1995) for radioimmuno-imaging.

Because the Abs were raised against the chelators and metal chelatecomplexes, they have remarkable specificity for the complex againstwhich they were originally raised. Indeed, the bsAbs of Boden et al.have specificity for single enantiomers of enantiomeric mixtures ofchelators and metal-chelate complexes.

Chelators such as those disclosed in U.S. Pat. No. 5,753,206, especiallythiosemi-carbazonylglyoxylcysteine(TscG-Cys) andthiosemicarbazinyl-acetylcysteine (TscA-Cys) chelators areadvantageously used to bind soft acid cations of Tc, Re, Bi and othertransition metals, lanthanides and actinides that are tightly bound tosoft base ligands, especially sulfur- or phosphorus-containing ligands.It can be useful to link more than one type of chelator to a peptide,e.g., a DTPA or similar chelator for, say In(III) cations, and athiol-containing chelator, e.g., TscG-Cys, for Tc cations. Becauseantibodies to a di-DTPA hapten are known (Barbet, U.S. Pat. No.5,256,395) and are readily coupled to a targeting antibody to form absAb, it is possible to use a peptide hapten with non-radioactive diDTPAchelates and another chelate for binding a radioisotope, in apretargeting protocol, for targeting the radioisotope. One example ofsuch a peptide is Ac-Lys(DTPA)-Tyr-Lys(DTPA)-Lys(TscG-Cys-)-NH₂. Thispeptide can be preloaded with In(III) and then labeled with 99-m-Tccations, the In(III) ions being preferentially chelated by the DTPA andthe Tc cations binding preferentially to the thiol-containing TscG-CysC.Other hard acid chelators such as NOTA, DOTA, TETA and the like can besubstituted for the DTPA groups, and Mabs specific to them can beproduced using analogous techniques to those used to generate theanti-di-DTPA Mab.

It will be appreciated that two different hard acid or soft acidchelators can be incorporated into the targetable construct, e.g., withdifferent chelate ring sizes, to bind preferentially to two differenthard acid or soft acid cations, due to the differing sizes of thecations, the geometries of the chelate rings and the preferred complexion structures of the cations. This will permit two different metals,one or both of which may be radioactive or useful for MRI enhancement,to be incorporated into a targetable construct for eventual capture by apre-targeted bsAb.

Chelators are coupled to the carrier portion of a targetable constructusing standard chemistries. For instance, excess2-(p-isothiocyanato)benzyl-DTPA is reacted with peptide NH₂ groups toform thiourea bonds between the p-isothiocyanate of the chelator and thefree 1-α and 6-ε-amino groups of the peptide, when a peptide is thetargetable construct. Alternatively, the bis-anhydride of DTPA can becoupled directly to a free amine group on the peptide. The desiredchelator-peptide is purified chromatographically and is ready for use asa metal binding agent. Similarly, DOTA is mono-activated at one carboxylgroup using a carbodiimide, and two DOTA units are coupled to thepeptide's free amino-groups or DOTA tri-t-butyl ester is activated witha carbodiimide and the DOTA units are coupled to the free amines on thepeptide. (The protecting groups are removed on cleavage from the resin.)Chelators bearing groups specifically reactive with thiols are used forreaction with peptides such asAc-Cys-D-Tyr-D-Trp-Gly-D-Cys-Gly-D-Tyr-D-Trp-NH₂. Such a chelator isexemplified by 2-(p-bromoacetamido)benzyl-DTPA, which may be used toalkylate the peptide's free thiol groups under mild, neutral conditions.

Chelator-peptide conjugates may be stored for long periods as solids.They may be metered into unit doses for metal-binding reactions, andstored as unit doses either as solids, aqueous or semi-aqueoussolutions, frozen solutions or lyophilized preparations. They may belabeled by well-known procedures. Typically, a hard acid cation isintroduced as a solution of a convenient salt, and is taken up by thehard acid chelator and possibly by the soft acid chelator. However,later addition of soft acid cations leads to binding thereof by the softacid chelator, displacing any hard acid cations which may be chelatedtherein. For example, even in the presence of an excess ofnon-radioactive InCl₃, labeling with ^(99m)Tc(V) glucoheptonate or withTc cations generated in situ with stannous chloride and Na99m-TcO₄proceeds quantitatively on the soft acid chelator. Other soft acidcations such as ¹⁸⁶Re, ¹⁸⁸Re, ²¹³Bi and divalent or trivalent cations ofMn, Co, Ni, Pb, Cu, Cd, Au, Fe, Ag (monovalent), Zn and Hg, especially⁶⁴Cu and ⁶⁷Cu, and the like, some of which are useful forradioimmunodiagnosis or radloimmunotherapy, can be loaded onto thecarrier peptide by analogous methods. Re cations also can be generatedin situ from perrhenate and stannous ions or a prereduced rheniumglucoheptonate or other transchelator can be used. Because reduction ofperrhenate requires more stannous ion (typically above 200 ug/mL finalconcentration) than is needed for the reduction of technetium, extracare needs to be taken to ensure that the higher levels of stannous iondo not reduce sensitive disulfide bonds such as those present indisulfide-cyclized peptides. A convenient way to prepare ReO metalcomplexes of the TscG-Cys-ligands is by reacting the peptide withReOCI₃(P(Ph₃)₂ but it is also possible to use other reduced species suchas ReO(ethylenediamine)₂.

Preferred chelators include NOTA, DOTA and Tscg and combinationsthereof. These chelators have been incorporated into a chelator-peptideconjugate motif as exemplified in the following constructs:

-   -   (a) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂    -   (b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂    -   (c) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂

The chelator-peptide conjugates (d) and (e), above, have been shown tobind ⁶⁸Ga and is thus useful in positron emission tomography (PET)applications.

Chelators are coupled to the linker moieties using standard chemistrieswhich are discussed more fully in the working Examples below. Briefly,the synthesis of the peptideAc-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys-)-NH₂ was accomplished by firstattaching Aloc-Lys(Fmoc)-OH to a Rink amide resin on the peptidesynthesizer. The protecting group abbreviations “Aloc” and “Fmoc” usedherein refer to the groups allyloxycarbonyl and fluorenylmethyloxycarbonyl. The Fmoc-Cys(Trt)-OH and TscG were then added to the sidechain of the lysine using standard Fmoc automated synthesis protocols toform the following peptide: Aloc-Lys(Tscg-Cys(Trt)-rink resin. The Alocgroup was then removed. The peptide synthesis was then continued on thesynthesizer to make the following peptide:(Lys(Aloc)-D-Tyr-Lys(Aloc)-Lys(Tscg-Cys(Trt)-)-rink resin. FollowingN-terminus acylation, and removal of the side chain Aloc protectinggroups. The resulting peptide was then treated with activatedN-trityl-HSG-OH until the resin gave a negative test for amines usingthe Kaiser test (Karacay et al., Bioconjugate Chem. 11:842-854, 2000).The synthesis of Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys-)-NH₂, as wellas the syntheses of DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; andDOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂ are described in greater detailbelow.

Chelator-peptide conjugates may be stored for long periods as solids.They may be metered into unit doses for metal-binding reactions, andstored as unit doses either as solids, aqueous or semi-aqueoussolutions, frozen solutions or lyophilized preparations. They may belabeled by well-known procedures. Typically, a hard acid cation isintroduced as a solution of a convenient salt, and is taken up by thehard acid chelator and possibly by the soft acid chelator. However,later addition of soft acid cations leads to binding thereof by the softacid chelator, displacing any hard acid cations which may be chelatedtherein. For example, even in the presence of an excess of cold¹¹¹InCl₃, labeling with 99m-Tc(V) glucoheptonate or with Tc cationsgenerated in situ with stannous chloride and Na99m-TcO₄ proceedsquantitatively on the soft acid chelator. Other soft acid cations suchas ¹⁸⁶Re, ¹⁸⁸Re, ²¹³Bi and divalent or trivalent cations of Mn, Co, Ni,Pb, Cu, Cd, Au, Fe, Ag (monovalent), Zn and Hg, especially ⁶⁴Cu and⁶⁷Cu, and the like, some of which are useful for radioimmunodiagnosis orradioimmunotherapy, can be loaded onto the linker peptide by analogousmethods. Re cations also can be generated in situ from perrhenate andstannous ions or a prereduced rhenium glucoheptonate or othertranschelator can be used. Because reduction of perrhenate requires morestannous ion (typically above 200 μg/mL final concentration) than isneeded for the reduction of Tc, extra care needs to be taken to ensurethat the higher levels of stannous ion do not reduce sensitive disulfidebonds such as those present in disulfide-cyclized peptides. Duringradiolabeling with rhenium, similar procedures are used as are used withthe Tc-99m. A preferred method for the preparation of ReO metalcomplexes of the Tscg-Cys-ligands is by reacting the peptide withReOCl₃(P(Ph₃)₂ but it is also possible to use other reduced species suchas ReO(ethylenediamine)₂.

V. Biologically Active Moieties

The targetable construct can be conjugated to or complexed with one ormore biologically active agents or moieties. The following arenon-limiting examples of biologically active moieties and agents.

One type of biologically active agent or moiety is an enzyme capable ofactivating a prodrug at the target site or improving the efficacy of anormal therapeutic by controlling the body's detoxification pathways.Suitable enzyme includes malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,α-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, α-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

Following administration of the bsAb, an enzyme conjugated to thecarrier is administered. After the enzyme is pre-targeted to the targetsite, a cytotoxic drug is injected, which is known to act at the targetsite, or a prodrug form thereof which is converted to the drug in situby the pre-targeted enzyme. The drug is one which is detoxified to forman intermediate of lower toxicity, most commonly a glucuronide, usingthe mammal's ordinary detoxification processes. The detoxifiedintermediate, e.g., the glucuronide, is reconverted to its more toxicform by the pre-targeted enzyme and thus has enhanced cytotoxicity atthe target site. This results in a recycling of the drug. Similarly, anadministered prodrug can be converted to an active drug through normalbiological processes. The pre-targeted enzyme improves the efficacy ofthe treatment by recycling the detoxified drug. This approach can beadopted for use with any enzyme-drug pair. Similar pre-targetingstrategies have been described in U.S. Application Ser. No. 60/101,039.Those methodologies are easily adaptable to the present invention andare hereby incorporated in their entirety by reference.

The enzyme-carrier conjugate can be mixed with the targeting bsAb priorto administration to the patient. After a sufficient time has passed forthe enzyme-carrier-bsAb conjugate to localize to the target site and forunbound conjugate to clear from circulation, a prodrug is administered.As discussed above, the prodrug is then converted to the drug in situ bythe pre-targeted enzyme.

Certain cytotoxic drugs that are useful for anticancer therapy arerelatively insoluble in serum. Some are also quite toxic in anunconjugated form, and their toxicity is considerably reduced byconversion to prodrugs. Conversion of a poorly soluble drug to a moresoluble conjugate, e.g., a glucuronide, an ester of a hydrophilic acidor an amide of a hydrophilic amine, will improve its solubility in theaqueous phase of serum and its ability to pass through venous, arterialor capillary cell walls and to reach the interstitial fluid bathing thetumor. Cleavage of the prodrug deposits the less soluble drug at thetarget site. Many examples of such prodrug-to-drug conversions aredisclosed in U.S. application Ser. No. 08/445,110.

Conversion of certain toxic substances such as aromatic or alicyclicalcohols, thiols, phenols and amines to glucuronides in the liver is thebody's method of detoxifying them and making them more easily excretedin the urine. One type of anti-tumor drug that can be converted to sucha substrate is epirubicin, a 4-epimer of doxorubicin (Adriamycin), whichis an anthracycline glycoside and has been shown to be a substrate forhuman beta-D-glucuronidase. See, e.g., Arcamone, Cancer Res. 45:5995,1985. Other analogues with fewer polar groups are expected to be morelipophilic and show greater promise for such an approach. Other drugs ortoxins with aromatic or alicyclic alcohol, thiol or amine groups arecandidates for such conjugate formation. These drugs, or other prodrugforms thereof, are suitable candidates for the site-specific enhancementmethods of the present invention.

The prodrug CPT-11 (irinotecan) is converted in vivo by carboxylesteraseto the active metabolite SN-38. SN-38 is a highly effective anti-tumoragent; however, therapeutic doses can not be administered to patientsdue to its toxicity. One application of the invention, therefore, is totarget such therapies to the tumor site using a bsAb specific for atumor-associated antigen and a hapten (e.g. di-DTPA) followed byinjection of a di-DTPA-carboxylesterase conjugate. Once a suitabletumor-to-background localization ratio has been achieved, the CPT-11 isgiven and the tumor-localized carboxylesterase serves to convert CPT-11to SN-38 at the tumor. Due to its poor solubility, the active SN-38 willremain in the vicinity of the tumor and, consequently, will exert aneffect on adjacent tumor cells that are negative for the antigen beingtargeted. This is a further advantage of the method. Modified forms ofcarboxylesterases have been described and are within the scope of theinvention. See, e.g., Potter et al., Cancer Res. 58:2646-2651 and3627-3632, 1998.

Etoposide is a widely used cancer drug that is detoxified to a majorextent by formation of its glucuronide and is within the scope of theinvention. See, e.g., Hande et al., Cancer Res. 48:1829-1834, 1988.Glucuronide conjugates can be prepared from cytotoxic drugs and can beinjected as therapeutics for tumors pre-targeted with mAb-glucuronidaseconjugates. See, e.g., Wang et al., Cancer Res. 52:4484-4491, 1992.Accordingly, such conjugates also can be used with the pre-targetingapproach described here. Similarly, designed prodrugs based onderivatives of daunomycin and doxorubicin have been described for usewith carboxylesterases and glucuronidases. See, e.g., Bakina et al., J.Med Chem. 40:4013-4018, 1997. Other examples of prodrug/enzyme pairsthat can be used within the present invention include, but are notlimited to, glucuronide prodrugs of hydroxy derivatives of phenolmustards and beta-glucuronidase; phenol mustards or CPT-II andcarboxypeptidase; methotrexate-substituted alpha-amino acids andcarboxypeptidase A; penicillin or cephalosporin conjugates of drugs suchas 6-mercaptopurine and doxorubicin and beta-lactamase; etoposidephosphate and alkaline phosphatase.

The enzyme capable of activating a prodrug at the target site orimproving the efficacy of a normal therapeutic by controlling the body'sdetoxification pathways may be conjugated to the hapten. Theenzyme-hapten conjugate is administered to the patient followingadministration of the pre-targeting bsAb and is directed to the targetsite. After the enzyme is localized at the target site, a cytotoxic drugis injected, which is known to act at the target site, or a prodrug formthereof which is converted to the drug in situ by the pre-targetedenzyme. As discussed above, the drug is one which is detoxified to forman intermediate of lower toxicity, most commonly a glucuronide, usingthe mammal's ordinary detoxification processes. The detoxifiedintermediate, e.g., the glucuronide, is reconverted to its more toxicform by the pre-targeted enzyme and thus has enhanced cytotoxicity atthe target site. This results in a recycling of the drug. Similarly, anadministered prodrug can be converted to an active drug through normalbiological processes. The pre-targeted enzyme improves the efficacy ofthe treatment by recycling the detoxified drug. This approach can beadopted for use with any enzyme-drug pair. In an alternative embodiment,the enzyme-hapten conjugate can be mixed with the targeting bsAb priorto administration to the patient. After a sufficient time has passed forthe enzyme-hapten-bsAb conjugate to localize to the target site and forunbound conjugate to clear from circulation, a prodrug is administered.As discussed above, the prodrug is then converted to the drug in situ bythe pre-targeted enzyme.

One type of biologically active agent or moiety is a prodrug. Thepre-targeting bsAb is administered to the patient and allowed tolocalize to the target and substantially clear circulation. At anappropriate later time, a targetable construct comprising a prodrug, forexample poly-glutamic acid (SN-38-ester)₁₀, is given, thereby localizingthe prodrug specifically at the tumor target. It is known that tumorshave increased amounts of enzymes released from intracellular sourcesdue to the high rate of lysis of cells within and around tumors. Apractitioner can capitalize on this fact by appropriately selectingprodrugs capable of being activated by these enzymes. For example,carboxylesterase activates the prodrug poly-glutamic acid(SN-38-ester)loby cleaving the ester bond of the poly-glutamic acid(SN-38-ester)₁₀ releasing large concentrations of free SN-38 at thetumor. Alternatively, the appropriate enzyme also can be targeted to thetumor site.

After cleavage from the targetable construct, the drug is internalizedby the tumor cells. Alternatively, the drug can be internalized as partof an intact complex by virtue of cross-linking at the target. Thetargetable construct can induce internalization of tumor-bound bsAb andthereby improve the efficacy of the treatment by causing higher levelsof the drug to be internalized.

A variety of carriers are well-suited for conjugation to prodrugs,including polyamino acids, such as polylysine, polyglutamic (E) andaspartic acids (D), including D-amino acid analogs of the same,co-polymers, such as poly(Lys-Glu) {poly[KE]}, advantageously from 1:10to 10:1. Copolymers based on amino acid mixtures such aspoly(Lys-Ala-Glu-Tyr) (KAEY; 5:6:2:1) can also be employed. Smallerpolymeric carriers of defined molecular weight can be produced bysolid-phase peptide synthesis techniques, readily producing polypeptidesof from 2-50 residues in chain length. A second advantage of this typeof reagent, other than precise structural definition, is the ability toplace single or any desired number of chemical handles at certain pointsin the chain. These can be used later for attachment of recognition andtherapeutic haptens at chosen levels of each moiety.

Poly(ethylene) glycol [PEG] has desirable in vivo properties for abi-specific antibody prodrug approach. Ester linkages between thehydroxyl group of SN-38 and both ends of a standard di-hydroxyl PEG canbe introduced by insertion of diacids such as succinic acid between theSN-38 and PEG hydroxyl groups, to generate species such asSN-38-O—CO(CH₂)₂CO—O-PEG-O—CO(CH₂)₂CO—OSN-38. The di-SN-38-PEG producedcan be considered as the shortest member of the class of SN-38-polymerprodrugs. The desirable in vivo properties of PEG derivatives and thelimited loading capacity due to their dimeric functionality led to thepreparation of PEG co-polymers having greater hapten-bearing capacitysuch as those described by Poiani et al. See, e.g., Poiani et al.,Bioconjugate Chem. 5:621-630, 1994. PEG derivatives are activated atboth ends as their bis(succinimidyl)carbonate derivatives andco-polymerized with multi-functional diamines such as lysine. Theproduct of such co-polymerization, containing(-Lys(COOH)—PEG-Lys(COOH)—PEG-)_(n) repeat units wherein the lysylcarboxyl group is not involved in the polymerization process, can beused for attachment of SN-38 residues. The SN-38 residues are reactedwith the free carboxyl groups to produce SN-38 esters of the(-Lys-(COOH)-PEG-Lys(COOH)-PEG-)_(n) chain.

Other synthetic polymers that can be used to carry recognition haptensand prodrugs include N-(2-hydroxypropyl)methacrylamide (HMPA)copolymers, poly(styrene-co-maleic acid/anhydride (SMA),poly(divinylether maleic anhydride) (DIVEMA), polyethyleneimine,ethoxylated polyethylene-imine, starburst dendrimers andpoly(N-vinylpyrrolidone) (PVP). As an example, DIVEMA polymer comprisedof multiple anhydride units is reacted with a limited amount of SN-38 toproduce a desired substitution ratio of drug on the polymer backbone.Remaining anhydride groups are opened under aqueous conditions toproduce free carboxylate groups. A limited number of the freecarboxylate groups are activated using standard water-soluble peptidecoupling agents, e.g. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC), and coupled to a recognition moiety bearing a freeamino group. An example of the latter is histamine, to which antibodieshave been raised in the past.

A variety of prodrugs can be conjugated to the carrier portion of thetargetable construct. The above exemplifications of polymer use areconcerned with SN-38, the active metabolite of the prodrug CPT-11(irinotecan). SN-38 has an aromatic hydroxyl group that was used in theabove descriptions to produce aryl esters susceptible to esterase-typeenzymes. Similarly the camptothecin analog topotecan, widely used inchemotherapy, has an available aromatic hydroxyl residue that can beused in a similar manner as described for SN-38, producingesterase-susceptible polymer-prodrugs.

Doxorubicin also contains aromatic hydroxyl groups that can be coupledto carboxylate-containing polymeric carriers using acid-catalyzedreactions similar to those described for the camptothecin family.Similarly, doxorubicin analogs like daunomycin, epirubicin andidarubicin can be coupled in the same manner. Doxorubicin and otherdrugs with amino ‘chemical handles’ active enough for chemical couplingto polymeric carriers can be effectively coupled to carrier moleculesvia these free amino groups in a number of ways. Polymers bearing freecarboxylate groups can be activated in situ (EDC) and the activatedpolymers mixed with doxorubicin to directly attach the drug to theside-chains of the polymer via amide bonds. Amino-containing drugs canalso be coupled to amino-pendant polymers by mixing commerciallyavailable and cleavable cross-linking agents, such as ethyleneglycobis(succinimidylsuccinate) (EGS, Pierce Chemical Co., Rockford,Ill.) or bis-[2-(succinimido-oxycarbonyloxy)ethyl]sulfone (BSOCOES,Molecular Biosciences, Huntsville, Ala.), to cross-link the two aminesas two amides after reaction with the bis(succinimidyl) ester groups.This is advantageous as these groups remain susceptible to enzymaticcleavage. For example, (doxorubicin-EGS)_(n)-poly-lysine remainssusceptible to enzymatic cleavage of the diester groups in the EGSlinking chain by enzymes such as esterases. Doxorubicin also can beconjugated to a variety of peptides, for example,HyBnK(DTPA)YK(DTPA)-NH₂, using established procedures(HyBn=p-H₂NNHC₆H₄CO₂H). See Kaneko et al., J. Bioconjug. Chem.2:133-141, 1991.

The therapeutic conjugate may comprise doxonibicin coupled to a carriercomprising amine residues and a chelating agent, such as DTPA, to form aDTPA-peptide-doxorubicin conjugate, wherein the DTPA forms therecognition moiety for a pretargeted bsMAb. Preferably, the carriercomprises a tyrosyl-lysine dipeptide, e.g., Tyr-Lys(DTPA)-NH₂, and morepreferably still it comprises Lys(DTPA)-Tyr-Lys(DTPA)-NH₂. Doxorubicinphenyl hydrazone conjugates to bis-DPTA containing peptides areparticularly desirable in a therapeutic context.

Methotrexate also has an available amino group for coupling to activatedcarboxylate-containing polymers, in a similar manner to that describedfor doxorubicin. It also has two glutamyl carboxyl groups (alpha andgamma) that can be activated for coupling to amino-group containingpolymers. The free carboxylate groups of methotrexate can be activatedin situ (EDC) and the activated drug mixed with an amino-containingpolymer to directly attach the drug to the side-chains of the polymervia amide bonds. Excess unreacted or cross-reacted drug is separatedreadily from the polymer-drug conjugate using size-exclusion orion-exchange chromatography.

Maytansinoids and calicheamicins (such as esperamycin) contain mixed di-and tri-sulfide bonds that can be cleaved to generate species with asingle thiol useful for chemical manipulation. The thiomaytensinoid orthioespera-mycin is first reacted with a cross-linking agent such as amaleimido-peptide that is susceptible to cleavage by peptidases. TheC-terminus of the peptide is then activated and coupled to anamino-containing polymer such as polylysine.

An immunomodulator, such as a cytokine, may also be conjugated to, orform an alternative or additional biologically active moiety. As usedherein, the term “immunomodulator” includes cytokines, stem cell growthfactors, lymphotoxins, such as tumor necrosis factor (TNF), andhematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1),IL-2, IL-3, IL-6, IL-10, IL-12, IL-18 and IL-21), colony stimulatingfactors (e.g., granulocyte-colony stimulating factor (G-CSF) andgranulocyte macrophage-colony stimulating factor (GM-CSF)), interferons(e.g., interferons-α, -β and -γ), the stem cell growth factor designated“S1 factor”, and erythropoietin and thrombopoietin. Examples of suitableimmunomodulator moieties include IL-2, IL-6, IL-10, IL-12, IL-18,interferon-γ, TNF-α, and the like. Alternatively, subjects can receiveinvention compositions and a separately administered cytokine, which canbe administered before, concurrently or after administration of theinvention compositions. The invention compositions may also beconjugated to the immunomodulator.

VI. Combination Therapy

The bi-specific antibody-directed delivery of therapeutics or prodrugpolymers to in vivo targets can be combined with bi-specific antibodydelivery of radionuclides, such that combination chemotherapy andradioimmunotherapy is achieved. Each therapy can be conjugated to thetargetable construct and administered simultaneously, or the nuclide canbe given as part of a first targetable construct and the drug given in alater step as part of a second targetable construct. In one simpleembodiment, a peptide containing a single prodrug and a single nuclideis constructed. For example, the tripeptide Ac-Glu-Gly-Lys-NH₂ can beused as a carrier portion of a targetable construct, whereby SN-38 isattached to the gamma glutamyl carboxyl group as an aryl ester, whilethe chelate DOTA is attached to the epsilon amino group as an amide, toproduce the complex Ac-Glu(SN-38)-Gly-Lys(DOTA)-NH₂. The DOTA chelatecan then be radiolabeled with various metals for imaging and therapypurposes including In-111, Y-90, Sm-153, Lu-177 and Zr-89. As themetal-DOTA complex may represent the recognizable hapten on thetargetable construct, the only requirement for the metal used as part ofthe DOTA complex is that the secondary recognition antibody also usedrecognizes that particular metal-DOTA complex at a sufficiently highaffinity. Generally, this affinity (log K_(a)) is between 6-11.Polymeric peptides such as poly[Glu(SN-38)₁₀-Lys(Y-90-DOTA)₂] can begiven as readily as the more chemically defined lower MW reagent above,and are indeed preferred. Also, triply substituted polymers can be used,such as poly[Glu(Sn-38)₁₀-Lys(Y-90-DOTA)_(n)(histamine-succinate)_(m),where n and m are integers, such that the recognition agent isindependent of the radioimmunotherapy agent. The prodrug is activated bycarboxylesterases present at the tumor site or by carboxylesterasestargeted to the site using a second targetable construct.

Alternatively, a combination therapy can be achieved by administeringthe chemotherapy and radioimmunotherapy agents in separate steps. Forexample, a patient expressing CEA-tumors is first administered bsAb withat least one arm which specifically binds CEA and at least one other armwhich specifically binds the targetable construct whose hapten is aconjugate of yttrium-DOTA. Later the patient is treated with atargetable construct comprising a conjugate ofyttrium-DOTA-beta-glucuronidase. After sufficient time for bsAb andenzyme localization and clearance, a second targetable construct,comprising Ac-Glu(SN-38)-Gly-Lys(Y-90-DOTA)-NH₂, is given. The secondtargetable construct localizes to the tumor by virtue of bsAb at thetumor that are not already bound to a first targetable construct. Firsttargetable constructs which are localized to the target site act on theAc-Glu(SN-38)-Gly-Lys(Y-90-DOTA)-NH₂ to liberate the free SN-38 drug.Localization of both the prodrug and its respective enzyme to the targetsite enhances the production of active drug by ensuring that the enzymeis not substrate limited. This embodiment constitutes a markedimprovement of current prodrug methodologies currently practiced in theart.

Another advantage of administering the prodrug-polymer in a later step,after the nuclide has been delivered as part of a previously giventargetable construct, is that the synergistic effects of radiation anddrug therapy can be manipulated and, therefore, maximized. It ishypothesized that tumors become more ‘leaky’ after RAIT due to radiationdamage. This can allow a polymer-prodrug to enter a tumor morecompletely and deeply. This results in improved chemotherapy.

Alternatively, the RAIT therapy agent can be attached to bsAb rather thetargetable construct. For example, an anti-CEA x anti-DTPA bsAbconjugated to Y-90-DOTA is administered first to a patient withCEA-expressing tumors. In this instance, advantage is taken of theselectivity of certain anti-chelate mabs in that an anti-indium-DTPAantibody do not bind to a yttrium-DOTA chelate. After theY-90-DOTA-anti-CEA x anti-indium-DTPA has maximized at the tumor andsubstantially cleared non-target tissue, a conjugate ofindium-DTPA-glucuronidase is injected and localized specifically to theCEA tumor sites. The patient is then injected with a polymer-prodrugsuch as poly(Glu)(SN-38)₁₀. The latter is cleaved selectively at thetumor to active monomeric SN-38, successfully combining chemotherapywith the previously administered RAIT.

It should also be noted that a bi-specific antibody or antibody fragmentcan be used in the present method, with at least one binding sitespecific to an antigen at a target site and at least one other bindingsite specific to an enzyme. Such an antibody can bind the enzyme priorto injection, thereby obviating the need to covalently conjugate theenzyme to the antibody, or it can be injected and localized at thetarget site and, after non-targeted antibody has substantially clearedfrom the circulatory system of the mammal, enzyme can be injected in anamount and by a route which enables a sufficient amount of the enzyme toreach the pre-targeted bsAb and bind to it to form an antibody-enzymeconjugate in situ.

VII. Kits

In accordance with yet another aspect of the present invention, thepresent invention provides a kit suitable for treating or identifyingdiseased tissues in a patient, comprising a bi-specific antibody orantibody fragment having at least one arm that specifically binds atargeted tissue and at least one other arm that specifically binds atargetable construct, a first targetable construct which comprises acarrier portion which comprises or bears at least one epitoperecognizable by the at least one other arm of the bi-specific antibodyor antibody fragment, and one or more conjugated therapeutic agents, orenzymes, and, optionally, a clearing composition useful for clearingnon-localized antibodies and antibody fragments.

A “clearing agent” is an agent that clears unbound targetable constructfrom circulation, thereby facilitating circulating moiety from apatient's body, removal from blood circulation, or inactivation thereofin circulation. Preferably, the clearing agent has physical properties,such as size, charge, configuration or combinations thereof, that limitclearing agent access to the population of target cells recognized by atargetable construct used in the same treatment protocol as the clearingagent. This enhancement may be further improved by the administration ofan anti-idiotypic clearing agent, such as an anti-idiotypic monoclonalantibody specific for the determinant of the targeting conjugate, whichbinds to the tumor site. The clearance effect may be further enhanced byusing a galactosylated clearing agent, because a galactosylated clearingagent is rapidly cleared through the liver.

When the first targetable construct comprises an enzyme, the kit mayoptionally contain a prodrug, when the enzyme is capable of convertingthe prodrug to a drug at the target site, a drug which is capable ofbeing detoxified in the patient to form an intermediate of lowertoxicity, when the enzyme is capable of reconverting the detoxifiedintermediate to a toxic form, and, therefore, of increasing the toxicityof the drug at the target site, or a prodrug which is activated in thepatient through natural processes and is subject to detoxification byconversion to an intermediate of lower toxicity, when the enzyme iscapable of reconverting the detoxified intermediate to a toxic form,and, therefore, of increasing the toxicity of the drug at the targetsite, or a second targetable construct which comprises a carrier portionwhich comprises or bears at least one epitope recognizable by the atleast one other arm of the bi-specific antibody or antibody fragment,and a prodrug, when the enzyme is capable of converting the prodrug to adrug at the target site. Instruments which facilitate identifying ortreating diseased tissue also can be included in the kit. Examplesinclude, but are not limited to application devices, such as syringes.

A therapeutic kit of the invention comprises any of the followingreagents and/or components in any combination.

1. One or more therapeutic agents.

2. If the therapeutic agent(s) are not formulated for delivery via thealimentary canal, which includes but is not limited to sublingualdelivery, a device capable of delivering the therapeutic agent throughsome other routes. One type of device for parenteral delivery is asyringe that is used to inject the therapeutic agent into the body of ananimal in need of the therapeutic agent. Inhalation devices may also beused.

b3. Separate containers, each of which comprises one or more reagents ofthe kit. In a preferred embodiment, the containers are vials containsterile, lyophilized formulations of a therapeutic composition that aresuitable for reconstitution. Other containers include, but are notlimited to, a pouch, tray, box, tube, or the like. Kit components may bepackaged and maintained sterilely within the containers.

4. Instructions to a person using a kit for its use. The instructionscan be present on one or more of the kit components, the kit packagingand/or a kit package insert. Such instructions include, by way ofnon-limiting example, instructions for use of the kit and its reagents,for reconstituting lyophilized reagents or otherwise preparing reagents.

A preferred kit of the present invention comprises the elements usefulfor performing an immunoassay. A kit of the present invention cancomprise one or more experimental samples (i.e., formulations of thepresent invention) and one or more control samples bound to at least onepre-packed dipstick or ELISA plate, and the necessary means fordetecting immunocomplex formation (e.g., labelled secondary antibodiesor other binding compounds and any necessary solutions needed to resolvesuch labels, as described in detail above) between antibodies containedin the bodily fluid of the animal being tested and the proteins bound tothe dipstick or ELISA plate. It is within the scope of the inventionthat the kit can comprise simply a formulation of the present inventionand that the detecting means can be provided in another way.

VIII. Characterization of Targetable Constructs and Complexes

Any of the following methodologies, as well as others known in the artand/or described in the Examples herein, can be used to examine one ormore attributes of a targetable construct or complex.

VIII.A. Affinity for Epitopes

Affinity can be either absolute or relative. By absolute affinity, it ismeant that the assay for affinity gives defined numerical determinationsof the affinity of one compound for another. Comparison of the affinityof the complex being tested to that of a reference compound whosebinding affinity is known allows for the determination of relativebinding affinity of the test ligand.

Whether absolute or relative, affinity of one molecule for another canbe measured by any method known in the art. By way of non-limitingexample, such methods include competition assays, surface plasmonresonance, half-maximal binding assays, competition assays, Scatchardanalysis, direct force techniques (Wong et al., Direct forcemeasurements of the streptavidin-biotin interaction, Biomol. Eng.16:45-55, 1999), and mass spectrometry (Downard, Contributions of massspectrometry to structural immunology, J. Mass Spectrom. 35:493-503,2000).

VIII.A.1. Absolute Affinity

As regards absolute affinity, “low affinity” refers to binding whereinthe association constant (Ka) between two molecules is about 10⁵ M to10⁷ M. “Moderate affinity” refers to binding wherein the associationconstant (Ka) between two molecules is at least about 10⁷ M to 10⁸ M.“High affinity” refers to a binding wherein the association constantbetween the two molecules is at least about 10⁸ M to about 10¹⁴ M, andpreferably about 10⁹ M to about 10¹⁴ M, more preferably about 10¹⁰ M toabout 10¹⁴ M, and most preferably greater than about 1014 M.

The dissociation constant, Kd, is an equilibrium constant for thedissociation of one species into two, e.g., the dissociation of acomplex of two or more molecules into its components, for example,dissociation of a substrate from an enzyme. Exemplary Kd values forcompositions of the present invention are from about 10⁻⁷ M (100 nM) toabout 10⁻¹² M (0.001 nM). The stability constant is an equilibriumconstant that expresses the propensity of a species to form from itscomponent parts. The larger the stability constant, the more stable isthe species. The stability constant (formation constant) is thereciprocal of the instability constant (dissociation constant).

The affinity of the complexes of the invention for a target epitope, orthe affinity of a bi-specific antibody for a carrier epitope, is drivenby non-covalent interactions. There are four main non-covalentattractive forces between molecules: (i) electrostatic forces, whichoccur between between oppositely charged molecules such as amino groupsand carboxylic groups; (ii) hydrogen bonds, which are formed whenhydrogen atoms are shared between electronegative atoms such as nitrogenand oxygen; (iii) Van der Waals forces, which are generated betweenelectron clouds around molecules oppositely polarized by neighboringatoms; and (iv) hydrophobic interactions, which are formed when water isexcluded from the interface allowing hydrophobic molecules to interactin a waterless environment.

Non-covalent interactions can, but rarely do, have the strength of acovalent linkage (i.e., a chemical bond). In some instances, theaffinity of the complexes of the invention for a target epitope,although driven by non-covalent interactions, is so high as to approachthe strength of a covalent bond. This provides for complexes that arevery stable relative to other complexes.

Preferably, the affinity of a targetable complex for its cognate targetepitope, and/or the affinity of a bsAb for the carrier epitopes of atargetable construct, is a Kd of about 100 nM to about 0.01 nM; morepreferably, greater than about 100 nM, or greater than about 10 nM; mostpreferably, greater than about 1 nM, or greater than about 0.1 nM.Typical Kd for target epitopes are from about 0.1 nM to 100 nM,preferably from about 0.1 nM to 10 nM, more preferably from about 0.5 nMto 5 nM, or about 1 nM.

In the invention, when multiple copies of a carrier epitope are presenton the targetable construct, the affinity of an antibody for its cognatecarrier epitope may be greater than the affinity of an antibody for afree carrier epitope or for a monovalent tragetable construct comprisingthe carrier epitope. Additionally or alternatively, a multivalenttargetable construct having x carrier epitopes has a greater affinityfor its target epitope than would x number of constructs. Put anotherway, the compositions of the invention provide for synergistic, ratherthan merely additive, binding effects.

VIII.A.2. Surface Plasmon Resonance

Binding parameters such as Kd may be measured using surface plasmonresonance on a chip, for example, with a BIAcore® chip coated withimmobilized binding components. Surface plasmon resonance is used tocharacterize the microscopic association and dissociation constants ofreaction between an antibody or antibody fragment and its ligand. Suchmethods are generally described in the following references which areincorporated herein by reference. (Vely et al., BIAcore analysis to testphosphopeptide-SH2 domain interactions, Meth. Mol. Biol. 121:313-21,2000; Liparoto et al., Biosensor analysis of the interleukin-2 receptorcomplex, J. Mol. Recog. 12:316-21, 1999; Lipschultz et al., Experimentaldesign for analysis of complex kinetics using surface plasmon resonance,Methods 20:310-8, 2000; Malmqvist., BIACORE: an affinity biosensorsystem for characterization of biomolecular interactions, Biochem. Soc.Transactions 27:33540, 1999; Alfthan, Surface plasmon resonancebiosensors as a tool in antibody engineering, Biosensors &Bioelectronics 13:653-63, 1998; Fivash et al., BIAcore formacromolecular interaction, Curr. Opin. Biotech. 9:97-101, 1998; Priceet al., Summary report on the ISOBM TD-4 Workshop: analysis of 56monoclonal antibodies against the MUC1 mucin, Tumour Biol. 19 Suppl1:1-20, 1998; Malmqvist et al., Biomolecular interaction analysis:affinity biosensor technologies for functional analysis of proteins,Curr. Opin. Chem. Biol. 1:378-83, 1997; O'Shannessy et al.,Interpretation of deviations from pseudo-first-order kinetic behavior inthe characterization of ligand binding by biosensor technology, Anal.Biochem. 236:275-83, 1996; Malmborg et al., BIAcore as a tool inantibody engineering, J. Immunol. Meth. 183:7-13, 1995; Van Regenmortel,Use of biosensors to characterize recombinant proteins, Dev. Biol.Standardization 83:143-51, 1994; O'Shannessy, Determination of kineticrate and equilibrium binding constants for macromolecular interactions:a critique of the surface plasmon resonance literature, Curr. Opin.Biotechnol. 5:65-71, 1994). Models using BIAcore to examine the bindingof fixed ligands to multivalent compounds have been described (Muller etal., Model and simulation of multivalent binding to fixed ligands, Anal.Biochem. 261:149-158, 1998).

BIAcore® uses the optical properties of surface plasmon resonance (SPR)to detect alterations in protein concentration bound within to a dextranmatrix lying on the surface of a gold/glass sensor chip interface, adextran biosensor matrix. In brief, proteins are covalently bound to thedextran matrix at a known concentration and a ligand for the protein(e.g., antibody) is injected through the dextran matrix. Near infraredlight, directed onto the opposite side of the sensor chip surface isreflected and also induces an evanescent wave in the gold film, which inturn, causes an intensity dip in the reflected light at a particularangle known as the resonance angle. If the refractive index of thesensor chip surface is altered (e.g., by ligand binding to the boundprotein) a shift occurs in the resonance angle. This angle shift can bemeasured and is expressed as resonance units (RUs) such that 1000 RUs isequivalent to a change in surface protein concentration of 1 ng/mm 2.These changes are displayed with respect to time along the y-axis of asensorgram, which depicts the association and dissociation of anybiological reaction.

Additional details may be found in Jonsson et al., Introducing abiosensor based technology for real-time biospecific interactionanalysis, Ann. Biol. Clin. 51:19-26, 1993; Jonsson et al., Real-timebiospecific interaction analysis using surface plasmon resonance and asensor chip technology, Biotechniques 11:620-627, 1991; Johnsson et al.,Comparison of methods for immobilization to carboxymethyl dextran sensorsurfaces by analysis of the specific activity of monoclonal antibodies,J. Mol. Recog. 8:125-131, 1995; and Johnsson, Immobilization of proteinsto a carboxymethyldextran-modified gold surface for biospecificinteraction analysis in surface plasmon resonance sensors, Anal.Biochem. 198:268-277, 1991; Karlsson et al., Kinetic analysis ofmonoclonal antibody-antigen interactions with a new biosensor basedanalytical system, J. Immunol. Meth. 145:229, 1991; Weinberger et al.,Recent trends in protein biochip technology, Pharmacogenomics 1:395-416,2000; Lipschultz et al., Experimental design for analysis of complexkinetics using surface plasmon resonance, Methods 20:310-8, 2000.

VIII.A.3. Relative Affinity

Affinity may also be defined in relative terms, e.g., by IC₅₀. In thecontext of affinity, the IC₅₀ of a compound is the concentration of thatcompound at which 50% of a reference ligand is displaced from a targetepitope in vitro or targeted tissue in vivo. When the target epitope isCEA, the reference ligand can be a complex comprising the[hMN]₂-[734scFv]₂ or [hMN^((1253A))]₂-[734scFv]₂ bi-specific antibody.Typically, IC₅₀ is determined by competitive ELISA.

VIII.B. Biodistribution and Clearing Characteristics

Methods of evaluating biodistribution patterns of targetable complexesare described in U.S. provisional Application Ser. No. 60/361,037 (Atty.Docket No. 018733-1037), which was filed Mar. 1, 2002 and is entitled“Bispecific antibody point mutations for enhancing rate of clearance.”This application is hereby incorporated in its entirety by reference.

Methods of evaluating clearing characteristics of targetable complexesare described in U.S. Provisional Application Ser. No. 60/361,037 (AttyDocket No. 018733-1037), which was filed Mar. 1, 2002 and is entitled“Bispecific antibody point mutations for enhancing rate of clearance.”This application is hereby incorporated in its entirety by reference.

VIII.C. Formation of Defined Species of Multimers

It is often the case that mixing several compounds that are capable ofbinding to each other results in a variety of multimers. For example,mixing binding compounds A and B can result in species of complexes suchas AB, (AB)₂, (AB)₃, (AB)₄, etc. A desirable attribute of some of thecomplexes of the invention is that the components thereof, when mixedtogether, predominately form a single type of multimer. In the case ofsome of the complexes of the invention, for example, dimeric complexesare predominately formed with little or no other species of multimersbeing present.

For example, mixing a targetable construct and one type bi-specificantibodies at relative concentrations ranging anywhere from about 10⁻⁹,10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³, 10⁻², 10⁻¹, 1, 10¹, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹ to about 10¹⁰ preferably results in a mixture inwhich greater than about 50% of the multimeric complexes have a definedstoichiometry of two molecules of the bi-specific antibody, and onemolecule of the targetable construct. Preferably ≧75% to about ≧85%,more preferably ≧95%, and most preferably ≧99% of the multimericcomplexes so formed have a defined stoichiometry of two molecules of thebi-specific antibody, and one molecule of the targetable construct.

VIII.D. Stability

VIII.D.1. Types of Stability

In general, two types of stability are of interest: chemical stabilityand conformational stability. Both types contribute to functionalstability: an agent may be chemically stable (i.e., resistant todegradation) but may not be biologically active if it does not have theproper conformation. The term “functional stability” refers to theamount of functional (biologically active) agent that is retained overtime.

Chemical stability is measured as is known in the art, e.g., bypreparing a mixture of labeled agent, incubating the mixture under agiven set of conditions (temperature, pH, etc.), and determining theamount of labeled agent remaining in samples taken at one or more timepoints. In addition to physiological conditions (see below), conditionsof interest may be those that influence the shelf-life of an agentand/or ease of manipulation thereof. The stability of an agent asregards a specific degradative molecule, e.g., in the case of proteins,proteases, can be determined in vitro using similar methodologies.

Conformational stability can be measured using a variety of techniquesknown in the art including, by way of non-limiting example, circulardichroism (CD), fluorescence, fluorescent energy transfer (FRET),fluorescent energy transfer confocal microscopy, nuclear magneticresonance (NMR) spectroscopy, neutron scattering, synchrotronradiolysis, mass spectrometry, and electrospray ionization massspectrometry. See, for example, van Mierlo and Steensma, Protein foldingand stability investigated by fluorescence, circular dichroism (CD), andnuclear magnetic resonance (NMR) spectroscopy: the flavodoxin story, J.Biotechnol. 79:281-98, 2000; Tehei et al., Fast dynamics of halophilicmalate dehydrogenase and BSA measured by neutron scattering undervarious solvent conditions influencing protein stability, Proc. Natl.Acad. Sci. USA. 98:14356-61, 2001; Maleknia and Downard, Unfolding ofapomyoglobin helices by synchrotron radiolysis and mass spectrometry,Eur. J. Biochem. 268:5578-88, 2001; Kim et al., Site-specific amidehydrogen/deuterium exchange in E. coli thioredoxins measured byelectrospray ionization mass spectrometry, J. Am. Chem. Soc. 123:9860-6,2001; Doig et al., Structure, stability and folding of the alpha-helix,Biochem. Soc. Symp. 68:95-110, 2001; Kolakowski and Konermann, Fromsmall-molecule reactions to protein folding: studying biochemicalkinetics by stopped-flow electrospray mass spectrometry, Anal. Biochem.292:107-14, 2001; Helfrich and Jones, High-throughput flow-injectiontechnique for stability sensing characterization of biomolecules insolution, Am. Laboratory 33:24-29, 2001; Hammarstrom et al., Proteincompactness measured by fluorescence resonance energy transfer: Humancarbonic anhydrase ii is considerably expanded by the interaction ofGroEL, J. Biol. Chem. 276:21765-75, 2001; Talaga et al., Dynamics andfolding of single two-stranded coiled-coil peptides studied byfluorescent energy transfer confocal microscopy, Proc. Natl. Acad. Sci.USA 97:13021-6, 2000; and Kumar and Nussinov, Review: How dothermophilic proteins deal with heat? Cell. Mol. Life Sci. 58:1216-1233,2001.

In addition to the conformational stability of individual components ofthe targetable complexes (e.g., targetable constructs and bsAbs), thestability of the targetable complexes per se is also a factor.Preferably, the targetable complexes of the invention are stable invitro and in vivo. That is, once the targetable constructs and bsAbs arecombined and form targetable complexes, the constructs and bsAbs havelittle tendency to dissociate from the complexes.

VIII.D.2. Stability Under Physiological Conditions

Another desirable attribute of a compound intended for in vivo use isstability particularly under physiological conditions. As those in theart will appreciate, what constitutes “physiological conditions” willvary, for example, depending on whether an in vivo or ex vivo state isunder consideration, the type of organism and its age, weight, health,sex, level of activity, metabolic state, etc. Parameters that vary invarious physiological conditions include, but are not limited to, thetype of solvent, pH, buffering capacity, the concentrations and types ofsalts and ions, temperature, and the like. In any event, it is wellwithin the skill of the ordinary artisan to define and determine whatparticular conditions exist for a given physiological state.

The stability of a compound can be expressed as the compound's half-lifein a body fluid such as, by way of non-limiting example, serum, blood,urine, lymph, plasma, interstitial fluid, bile, gastric juices and thelike. By way of non-limiting example, stability can be measured andexpressed as the in vivo or in vitro half-life of a compound in serum orblood.

For example, serum half-life is a time point at which half of theadministered amount of targeting protein or conjugate thereof remains inthe serum. Serum determinations over a series of time points cangenerate a curve which is useful for determining whole body exposure toan agent.

IX. Biosensors

IX.A. Biosensors in General

The present invention is directed to a device that comprises a sensoradapted to detect one or more specific health and/or nutrition markersin a subject or in the environment. The device may also signal thecaretaker, the subject, or an actuator of the occurrence. The sensorcomprises a biosensor. As used herein, the term “biosensor” is definedas a component comprising one or more binding moities being adapted todetect a ligand found in one or more target pathogenic microorganisms orrelated biomolecules.

Generally, biosensors function by providing a means of specificallybinding, and therefore detecting, a target biologically active analyte.In this way, the biosensor is highly selective, even when presented witha mixture of many chemical and biological entities. Often the targetbiological analyte is a minor component of a complex mixture comprisinga multiplicity of biological and other components. Thus, in manybiosensor applications, detection of target analytes occurs in theparts-per-billion, parts-per-trillion, or even lower ranges levels.

IX.B. Biosensor Design

The biosensor of the present invention may comprise a bio-recognitionelement, or molecular recognition element, that provides the highlyspecific binding or detection selectivity for a particular analyte. Thebio-recognition element or system is often an antibody. In a biosensorof the invention, the bio-recognition element, or system, is atargetable complex comprising bsAbs. The bio-recognition element isresponsible for the selective recognition of the analyte and thephysico-chemical signal that provides the basis for the output signal.

Biocatalytic and bioaffinity biosensor systems are described in moredetail in J. Chromatography 510:347-354, 1990, and in the Kirk-OthmerEncyclopedia of Chemical Technology, 4.sup.th ed. (1992), John Wiley &Sons, NY, each of which is incorporated by reference herein.

The biosensors of the present invention may detect biologically activeanalytes related to impending (i.e., future presentation of symptoms islikely) or current human systemic disease states, including, but notlimited to, pathogenic bacteria, parasites (e.g., any stage of the lifecycle, including eggs or portions thereof, cysts, or mature organisms),viruses, fungi, antibodies to pathogens, and/or microbially producedtoxins. Additionally, the biosensor may target biologically activeanalytes related to impending or current localized health issues, suchas stress proteins (e.g., cytokines) and interleukin 1-alpha that mayprecede the clinical presentation of skin irritation or inflammation. Insome embodiments, the biosensor functions as a proactive sensor,detecting and signaling the subject, a caretaker or medical personnel ofthe impending condition prior to the presentation of clinical symptoms.This allows time to administer prophylactic or remedial treatments tothe subject which can significantly reduce, if not prevent, the severityand duration of the symptoms. Further, the sensor, by detecting thepresence of a target biological analyte in a sample from the subject,may detect residual contamination on a surface, such as skin orenvironmental surface, in contact with the biosensor, and provide andappropriate signal.

The physico-chemical signal generated by the bio-recognition element orelements may be communicated visually to the caretaker or medicalpersonnel (i.e., via a color change visible to the human eye). Otherembodiments may produce optical signals, which may require otherinstrumentation to enhance the signal. These include flourescence,bioluminesence, total internal reflectance resonance, surface plasmonresonance, Raman methods and other laser-based methods, such as LED orlaser diode sensors. Exemplary surface plasmon resonance biosensors areavailable as IBIS I and IBIS II from XanTec Analysensysteme of Muenster,Germany, which may comprise bioconjugate surfaces as bio-recognitionelements. Alternatively, the signal may be processed via an associatedtransducer which, for example, may produce an electrical signal (e.g.,current, potential, inductance, or impedance) that may be displayed(e.g., on a readout such as an LED or LCD display) or which triggers anaudible or tactile (e.g., vibration) signal or which may trigger anactuator, as described herein. The signal may be qualitative (e.g.,indicating the presence of the target biological analyte) orquantitative (i.e., a measurement of the amount or concentration of thetarget biological analyte). In such embodiments, the transducer mayoptionally produce an optical, thermal or acoustic signal. In any event,the signal may also be durable (i.e., stable and readable over a lengthof time typically at least of the same magnitude as the usage life ofthe device) or transient (i.e., registering a real-time measurement).Additionally, the signal may be transmitted to a remote indicator site(e.g., via a wire, or transmitter, such as an infrared or rftransmitter) including other locations within or on the device or remotedevices. Further, the sensor, or any of its components, may be adaptedto detect and/or signal only concentrations of the target biologicalanalyte above a predefined threshold level (e.g., in cases wherein thetarget biological analyte is normally present in the bodily waste orwhen the concentration of the analyte is below a known “danger” level).

The target analytes that the biosensors of the present invention areadapted to detect may also be viruses. An exemplary biosensor adapted todetect HIV is described in U.S. Pat. Nos. 5,830,341 and 5,795,453,referenced above. The disclosure of each of these patents isincorporated by reference herein. Biosensors are adopted to use indifferent tissues; see, e.g., U.S. Pat. No. 6,342,037; and usingdifferent binding molecules, see, e.g., U.S. Pat. No. 6,329,160.

When the targetable complexes of the invention are incorporated into abiosensor, they may be immobilized in the biosensor by techniques knownin the art such as entrapment, adsorption, crosslinking, encapsulation,covalent attachment, any combination thereof, or the like. Further, theimmobilization can be carried out on many different substrates such asknown the art. In certain preferred embodiments, the immobilizationsubstrate may be selected from the group of polymer-based materials,hydrogels, tissues, nonwoven materials or woven materials.

In certain embodiments, biosensor embodiments, may comprise, be disposedon, or be operatively associated with a microchip, such as a siliconchip, MEMs (i.e., micro electromechanical system) device, or anintegrated circuit. Microchip-based biosensors may be known as“biochips.” Regardless of the type of sensor, the microchip may comprisea multiplicity of sensor components having similar or differentsensitivities, kinetics, and/or target analytes (i.e., markers) in anarray adapted to detect differing levels or combinations of theanalyte(s). Further, each sensor in such an array may provide adifferent type of signal, including those types disclosed herein, andmay be associated with different actuators and/or controllers. Also,each sensor in an array may operate independently or in association with(e.g., in parallel, combination, or series) any number of other sensorsin the array.

A biosensor of the invention may comprise a detectable compound thatproduces a signal once analytes are bound. See, by way of non-limitingexample, Billinton et al., Development of a green fluorescent proteinreporter for a yeast genotoxicity biosensor, Biosensors & Bioelectronics13:831-838, 1998. A biosensor according to the invention may usemicrobalance sensor systems (Hengerer et al., Determination of phageantibody affinities to antigen by a microbalance sensor system,BioTechniques 26:956-964, 1999).

X. Target Antigens and Epitopes

A target epitope is comprised within, displayed by and/or released fromtargeted tissues of a subject, samples or cell cultures thereof. Asample may be a bodily tissue or fluid tissue and may be within asubject, or biopsied or removed from a subject, or a whole or anyportion of a bodily organ. Additionally, the tissue may be “sample” inthat the tissue is recently removed from a subject without anypreservation steps between the excision and the methods of the currentinvention. The tissue may also have been preserved by such standardtissue preparation techniques including, but not limited to, freezing,quick freezing, paraffin embedding and tissue fixation, prior toapplication of the methods of the current invention.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to humans andother primates, rodents (e.g., mice, rats, and guinea pigs), lagamorphs(e.g., rabbits), bovines (e.g., cattle), ovines (e.g., sheep), caprines(e.g., goats), porcines (e.g., swine), equines (e.g., horses), canines(e.g., dogs), felines (e.g., cats), domestic fowl (e.g., chickens,turkeys, ducks, geese, other gallinaceous birds, etc.), as well as feralor wild animals, including, but not limited to, such animals asungulates (e.g., deer), bear, fish, lagamorphs, rodents, birds, etc. Itis not intended that the term be limited to a particular age or sex.Thus, adult and newborn subjects, as well as fetuses, whether male orfemale, are encompassed by the term. However, the preferred species foruse of this technology is Homo sapiens, and the next preferred use is indomestic pets, such as horses, dogs, and cats.

By “displayed” it is meant that a portion of the membrane protein ispresent on the surface of a cell, tissue and/or organ, and is thus incontact with the external environment of the cell, tissue or organ. Atarget epitope may be associated with a disease including but notlimited to cancers and pathogenic infections.

X.A. Antigens and Epitopes Associated with Hyperproliferative Diseases

The mutant bispecific antibodies used in the present invention arespecific to a variety of cell surface or intracellular antigensassociated with hyperproliferative diseases. Normal tissue homeostasisis achieved by an intricate balance between the rate of cellproliferation and cell death. Disruption of this balance either byincreasing the rate of cell proliferation or decreasing the rate of celldeath can result in the abnormal growth of cells and is thought to be amajor event in the development of cancer and other hyperproliferativediseases. A “hyperproliferative disease” is one in which cells have anabnormally high rate of cell division and/or an abnormally low rate ofnecrosis and/or apoptosis. Non-limiting examples include tumorigenesis;tumor progression; cancers, such as leukemia, solid tumors andmetastases; psoriasis; benign hyperproliferative diseases, such asbenign prostatic hypertrophy, benign hyperplasia of the skin, andhemangiomas; chronic inflammatory proliferative diseases, such aspsoriasis and rheumatoid arthritis; proliferative ocular disorders, suchas diabetic retinopathy and macular degeneration; and proliferativecardiovascular diseases, such as restenosis. Restenosis, characterizedby the regrowth of smooth muscle cells into the lumen of blood vesselsfollowing angioplasty or other arterial damage, is a frequent andrecurring problem in the long term success of angioplasty, and alsooccurs after arterial reconstructions, atherectomy, stent implantation,and laser angioplasty.

These antigens may be substances produced by, e.g., the tumor or may besubstances which accumulate at a tumor site, on tumor cell surfaces orwithin tumor cells, whether in the cytoplasm, the nucleus or in variousorganelles or subcellular structures, including cell-surface orintracellular receptors. Among such tumor-associated markers are thosedisclosed, but not intended to be limiting, by Herberman,Immunodiagnosis of Cancer, in Fleisher ed., The Clinical Biochemistry ofCancer, page 347 (American Association of Clinical Chemists, 1979) andin U.S. Pat. Nos. 4,150,149; 4,361,544; and 4,444,744.

Tumor-associated markers have been categorized by Herberman, supra, in anumber of categories including oncofetal antigens, placental antigens,oncogenic or tumor virus associated antigens, tissue associatedantigens, organ associated antigens, ectopic hormones and normalantigens or variants thereof. Occasionally, a sub-unit of atumor-associated marker is advantageously used to raise antibodieshaving higher tumor-specificity, e.g., the beta-subunit of humanchorionic gonadotropin (HCG) or the gamma region of carcino embryonicantigen (CEA), which stimulate the production of antibodies having agreatly reduced cross-reactivity to non-tumor substances as disclosed inU.S. Pat. Nos. 4,361,644 and 4,444,744.

Examples, which are non-limiting, of suitable tumor-associated markersor receptors, include the B-cell complex structures (e.g., CD19, CD20,CD21, CD22, CD23, CD80), other receptors expressed on hematopoietic andcertain solid tumors (e.g., CD15, CD33, CD45, NCA90, NCA95, CD74,HLA-DR), and tumor-associated markers expressed on diverse cancers(e.g., carcinoembryonic antigen, Le(y), MUC-1, MUC-2, MUC-3, MUC-4,Tag-72 [B72.3 and CC49 constituting the antibodies against Tag-72],EGP-1, EGP-2, the antigen specific for A33 antibody, PSA, PSMA, EGFR,HER2/neu, PAM-4, AFP, HCG and its subunits, melanoma-associated antigens(e.g., S100), glioma-associated antigens, ovarian cancer-associatedantigens, etc.), as well as target molecules expressed by thevasculature of the tumors (tumor angiogenesis markers, usually producedby the vascular endothelium), such as VEGF and tenascin (the latter inbrain tumors, for example), and also to oncogene-associated markers,such as p53. Other tumor-associated antigens include, but are notlimited to A3, BrE3, CD1, CD1a, CD3, CD5, CD15, CD25, CD30, CD33, CD45,CD79a, CSAp, EGP-1, EGP-2, Ep-CAM, Ba 733, KC4, KS-1, KS1-4, MAGE, RS5,IL-6, insulin growth factor-1 (IGF-1), Tn antigen, Thomson-Friedenreichantigens, tumor necrosis antigens, 17-1A, an angiogenesis marker, acytokine, an immunomodulator, an oncogene marker (e.g., p53), and anoncogene product. In addition to the exemplary antibodies to suchantigens disclosed herein, antibodies to these antigens are known in theart (see, for example, Kim et al., Expression and Characterization of aRecombinant Fab Fragment Derived from an Anti-Human alpha-FetoproteinMonoclonal Antibody, Mol. Cells 11: 158-163, 2001; and Haisma et al.,Construction and characterization of a fusion protein of single-chainanti-CD40 antibody and human

-glucuronidase for antibody-directed enzyme prodrug therapy, Blood92:184-190, 1998.

Another marker of interest is transmembrane activator andCAML-interactor (TACI). See Yu et al., Nat. Immunol. 1:252-256, 2000.Briefly, TACI is a marker for B-cell malignancies (e.g., lymphoma).Further it is known that TACI and B cell maturation antigen (BCMA) arebound by the tumor necrosis factor homolog a proliferation-inducingligand (APRIL). APRIL stimulates in vivo proliferation of primary B andT cells and increases spleen weight due to accumulation of B cells invivo. APRIL also competes with TALL-I (also called BLyS or BAFF) forreceptor binding. Soluble BCMA and TACI specifically prevent binding ofAPRIL and block APRIL-stimulated proliferation of primary B cells.BCMA-Fc also inhibits production of antibodies against keyhole limpethemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-Isignaling via BCMA and/or TACI are required for generation of humoralimmunity. Thus, APRIL-TALL-I and BCMA-TACI form a two ligand-tworeceptor pathway involved in stimulation of B and T cell function.

Tumor-specific antigens (TSAs), tumor-associated differentiationantigens (TADAs) and other antigens associated with cancers and otherhyperproliferative diseases also include, but are not limited to, C1IAC, a human cancer associated protein (U.S. Pat. No. 4,132,769); theCA125 antigen, an antigen associated with cystadenocarcinoma of theovary, (Hanisch et al., Carbohydr. Res. 178:29-47, 1988; U.S. Pat. No.4,921,790); CEA (carcinembryonic antigen), an antigen present on manyadenocarcinomas (Horig et al., Strategies for cancer therapy usingcarcinembryonic antigen vaccines, Expert Reviews in Molecular Medicine,http://www-ermm.cbcu.cam.ac.uk: 1, 2000); CORA (carcinoma ororosomucoid-related antigen) described by Toth et al. (U.S. Pat. No.4,914,021); DF3 antigen from human breast carcinoma (U.S. Pat. Nos.4,963,484 and 5,053,489); DU-PAN-2, a pancreatic carcinoma antigen (Lanet al., Cancer Res. 45:305-310, 1985); HCA, a human carcinoma antigen(U.S. Pat. No. 5,693,763); Her2, a breast cancer antigen (Fendly et al.,The Extracellular Domain of HER2/neu Is a Potential Immunogen for ActiveSpecific Immunotherapy of Breast Cancer, J. Biol. Resp. Modifiers9:449-455, 1990); MSA, a breast carcinoma glycoprotein (Tjandra et al.,Br. J. Surg. 75:811-817, 1988); MFGM, a breast carcinoma antigen (Ishidaet al., Tumor Biol. 10: 12-22, 1989); PSA, prostrate specific antigen(Nadji et al., Prostatic-specific-antigen, Cancer 48:1229-1232, 1981);STEAP (six transmembrane epithelial antigens of the prostate) proteins(U.S. Pat. No. 6,329,503); TAG-72, a breast carcinoma glycoprotein(Kjeldsen et al., Cancer Res. 48:2214-2220, 1988); YH206, a lungcarcinoma antigen (Hinoda et al., Cancer J. 42:653-658, 1988); the p97antigen of human melanoma (Estin et al., Recombinant Vaccinia VirusVaccine Against the Human Melanoma Antigen p97 for Use in Immunotherapy,Proc. Natl. Acad. Sci. USA 85:1052-1056, 1988); and the melanomaspecific antigen described in U.S. Pat. No. 6,025,191).

X.B. Antigens from Pathogens

X.B.1. Viruses

By way of non-limiting example, pathogens include viruses including, butnot limited to, hepatitis type A, hepatitis type B, hepatitis type C,influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpessimplex type II (HSV-11), rinderpest, rhinovirous, echovirus, rabiesvirus, Ebola virus, rotavirus, respiratory syncytial virus, papillomavirus, papova virus, cytomegalovirus (CMV), echinovirus, arbovirus,huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus,polio virus, human immunodeficiency virus type I (HIV-I) and humanimmunodeficiency virus type II (HIV-I), Sendai virus, feline leukemiavirus, Reovirus, poliovirus, human serum parvo-like virus, simian virus40 (SV40), respiratory syncytial virus (RSV), mouse mammary tumor virus(MMTV), Varicella-Zoster virus, Dengue virus, rubella virus, measlesvirus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus,murine leukemia virus, vesicular stomatitis virus (VSV), smallpox(Variola virus), Sindbis virus, lymphocytic choriomeningitis virus,Rinderpest virus, wart virus and blue tongue virus.

X.B.2. Intracellular Pathogens

By way of non-limiting example, pathogens include intracellularobligates, including but not limited to Chlamydia sp., Rickettsia sp.,intracellular protozoa, including but not limited to, species ofLeishmania, Kokzidioa, and Trypanosoma, including without limitationintracellular spirochetes, including but not limited to, Borreliaburgdorfei, the causative agent of Lyme disease; and species ofPlasmodia, sporozoan obligate intracellular parasites of liver and redblood cells, including but not limited to P. falciparum, the causativeagent of malaria, Trypanosoma brucei, a hemoflagellate causing sleepingsickness, and Trypanosoma cruzi, the cause of Chagas disease. Forreviews of the immunology of such pathogens, see Blackman, Proteasesinvolved in erythrocyte invasion by the malaria parasite: function andpotential as chemotherapeutic targets, Curr Drug Targets 1:59-83, 2000;Kosma, Chlamydial lipopolysaccharide, Biochim. Biophys. Acta.1455:387-402, 1999; Casadevall, Antibody-mediated protection againstintracellular pathogens, Trends Microbiol. 6:102-7, 1998; Hoffman andFranke, Inducing protective immune responses against the sporozoite andliver stages of Plasmodium, Immunol. Lett. 41:89-94, 1994; Keusch,Immune responses in parasitic diseases. Part A: general concepts, Rev.Infect. Dis. 4:751-5, 1982; and Colli and Alves, Relevant glycoproteinson the surface of Trypanosoma cruzi.

X.B.3. Bacteria

Bacterial pathogens include, but are not limited to, Streptococcusagalactiae, Legionella pneumophilia, Streptococcus pyogenes, Escherichiacoli, Salmonella typhimurium, Neisseria gonorrhoeae, Neisseriameningitidis, Pneumococcus sp., Hemophilis influenzae B, Yersina pestis,Mycobacteria sp. including by way of non-limiting example Mycobacteriumleprae and Mycobacterium tuberculosis, Treponema pallidum, Pseudomonasaeruginosa, Francisella tularensis, Brucella sp. including Brucellaabortus, Bacillus anthracis including Anthrax spores, Clostridiumbotulinum including Botulism toxin, and Clostridium tetani includingTetanus toxin). See U.S. Pat. No. 5,332,567.

X.B.4. Pathogenic Fungi

Fungal pathogens include, but are not limited to, Candida sp.,Aspergillus sp., Mucor sp., Rhizopus sp., Fusarium sp., Penicilliummarneffei and Microsporum. Trichophyton mentagrophytes, Candidaalbicans, Histoplasma capsulatum, Blastomyces dermatitidis, andCoccidioides immitis are fungal pathogens of particular interest.

XI. Antibodies

The Fvs of the invention constructs are derived from an antibody andspecifically bind a targeted tissue. Exemplary Fvs are derived fromanti-CD20 antibodies, such as those described in U.S. ProvisionalApplication Ser. No. 60/356,132, entitled “Anti-CD20 Antibodies AndFusion Proteins Thereof And Methods Of Use”, Attorney Docket No.18733-1073, filed Feb. 14, 2002 (the contents of which are incorporatedby reference herein in their entirety) and hMN-14 antibodies, such asthose disclosed in U.S. Pat. No. 5,874,540 (the contents of which areincorporated by reference herein in their entirety), which is a ClassIII anti-carcinoembryonic antigen antibody (anti-CEA antibody).

The Fvs can be from murine antibodies, cdr-grafted (humanized)antibodies, or human antibodies. The Fvs can be derived from humanmonoclonal antibodies, transgenic mice with human Fv-libraries, orphage/ribosome human IgG libraries.

When the Fvs are derived from CDR-grafted antibodies, appropriatevariable region framework sequences may be used having regard to theclass or type of the donor antibody from which the antigen bindingregions are derived. Preferably, the type of human framework used is ofthe same or similar class or type as the donor antibody. Advantageously,the framework is chosen to maximize or optimize homology with the donorantibody sequence, particularly at positions spatially close to oradjacent the CDRs. Examples of human frameworks which may be used toconstruct CDR-grafted antibodies are LAY, POM, TUR, TEI, KOL, NEWM, REIand EU. KOL and NEWM and are suitable for heavy chain construction. REIis suitable for light chain construction and EU is suitable for bothheavy chain and light chain construction.

The light or heavy chain variable regions of the CDR-grafted antibodiesmay be fused to human light or heavy chain constant domains asappropriate, (the term “heavy chain constant domains” as used herein isto be understood to include hinge regions unless specified otherwise).The human constant domains of the CDR-grafted antibodies, where present,may be selected having regard to the proposed function of the antibody,in particular, the effector functions which may be required. Forexample, IgG1 and IgG3 isotype domains may be used when the CDR-graftedantibody is intended for therapeutic purposes and antibody effectorfunctions are required. Alternatively, IgG2 and IgG4 isotype domains maybe used when the CDR-grafted antibody is intended for purposes for whichantibody effector functions are not required, e.g. for imaging,diagnostic or cytotoxic targeting purposes. Light chain human constantdomains which may be fused to the light chain variable region includehuman Lambda or, especially, human Kappa chains.

Antibodies may further contain desirable mutations, e.g., mutationsfacilitating clearance of antibody constructs. A mutation may encompass,for example, a “conservative” change, wherein a substituted amino hassimilar structural or chemical properties, such as charge or size (e.g.,replacement of leucine with isoleucine). A mutation also encompasses,for example, a “non-conservative” change (e.g., replacement of a glycinewith a tryptophan).

The scFv component of the bi-specific mutant antibody specifically bindsa targetable construct. The use of any scFv component is contemplated bythe present invention. Preferred scFv components are 679 scFv (derivedfrom a murine anti-HSG) and 734scFv (derived from a murine anti-diDTPA).The scFv can be murine, cdr-grafted (humanized) or human.

The light or heavy chain variable regions of the CDR-grafted antibodiesmay be fused to human light or heavy chain constant domains asappropriate, (the term “heavy chain constant domains” as used herein areto be understood to include hinge regions unless specified otherwise).The human constant domains of the CDR-grafted antibodies, where present,may be selected having regard to the proposed function of the antibody,in particular the effector functions which may be required. For example,IgG1 and IgG3 isotype domains may be used when the CDR-grafted antibodyis intended for therapeutic purposes and antibody effector functions arerequired. Alternatively, IgG2 and IgG4 isotype domains may be used whenthe CDR-grafted antibody is intended for purposes for which antibodyeffector functions are not required, e.g. for imaging, diagnostic orcytotoxic targeting purposes. Light chain human constant domains whichmay be fused to the light chain variable region include human Lambda or,especially, human Kappa chains.

The murine monoclonal antibody designated 679 (an IgG1, K) binds withhigh affinity to molecules containing the tri-peptide moiety histaminesuccinyl glycyl (HSG) (Morel et al., Mol. Immunol. 27:995-1000, 1990).The nucleotide sequence pertaining to the variable domains (V_(H) andV_(K)) of 679 has been determined (Qu et al, unpublished results). V_(K)is one of two isotypes of the antibody light chains, V_(L). The functionof the two isotypes is identical. 679 can be humanized or fully human tohelp avoid an adverse response to the murine antibody.

hMN14 is a humanized monoclonal antibody that binds specifically to CEA(Shevitz et al., J. Nucl. Med. 34, 217P, 1993; U.S. Pat. No. 6,254,868).While the original Mabs were murine, humanized antibody reagents are nowutilized to reduce the human anti-mouse antibody response. The variableregions of this antibody were engineered into an expression construct(hMN14-scFv-L5). A preferred mutant hMN14 is hMN-14IgG^(1253A) whereinamino acid residue 253 is changed from isoleucine to alanine.

734 is a murine monoclonal antibody designated that binds with highaffinity to the metal-chelate complex indium-DTPA(diethylenetriamine-pentaacetic acid).

Single light chain and two heavy chain variable region sequencesencoding the humanized anti-hCD20 (hA20) antibody were designed andconstructed, as in U.S. Provisional Application Ser. No. 60/356,132,entitled “Anti-CD20 Antibodies And Fusion Proteins Thereof And MethodsOf Use”, Attorney Docket No. 18733-1073, filed Feb. 14, 2002, and U.S.application Ser. No. 10/366,709, filed Feb. 14, 2003 (the contents ofeach of which are incorporated by reference herein in their entirety).ha20 contains the V_(H) and V_(K) genes of A20, an anti-CD20 antibody,obtained by RT-PCR using the primer pairs VH1BACK/VH1FOR andVK1BACK/VK1FOR, respectively Orlandi et al., Proc. Natl. Acad. Sci. USA86: 3833, 1989. Human REI framework sequences were used for V_(K), and acombination of EU and NEWM framework sequences were used for V_(H).There are a number of amino acid changes in each chain outside of theCDR regions when compared to the starting human antibody frameworks. Theheavy chain of hA20, hA20V_(H)1, contains nine changes, while hA20V_(H)2contains three changes from the human EU frameworks. hA20V_(H)2 ispreferred because it contains more amino acids from the human antibodyframework region than hA20V_(H)1. The light chain of hA20, hA20V_(K),contains seven amino acid changes from the REI framework.

The hLL-2 antibody is a humanized antibody prepared by combining the CDRregions of murine LL-2 antibody (mLL-2) with variable region frameworksequences obtained from human antibodies. The sequence of the heavy andlight chain variable regions of hLL-2 are shown in FIG. 1 of U.S. Pat.No. 5,789,554. As shown in that figure, the kappa light chain of hLL-2contains the four light chain CDR regions from mLL-2 and the fourframework regions of human antibody REI. The heavy chain of hLL-2contains the three heavy chain CDRs from mLL-2 combined with threeframework regions from human antibody EU, together with a fourthframework region from human antibody NEWM.

XI.A. Definitions

The term “antibody” is meant to encompass an immunoglobulin moleculeobtained by in vitro or in vivo generation of an immunogenic response,and includes both polyclonal, antipeptide and monoclonal antibodies. Theterm “antibody” also includes genetically engineered antibodies and/orantibodies produced by recombinant DNA techniques and “humanized”antibodies. As described below, humanized and even fully humanantibodies can be produced by phage display, gene and chromosometransfection methods, as well as by other means.

An “immunogenic response” or “antigenic response” is one that results inthe production of antibodies directed to a compound after theappropriate cells have been contacted therewith. The compound that isused to elicit an immunogenic response is referred to as an immunogen orantigen. The antibodies produced in the immunogenic responsespecifically bind the immunogen used to elicit the response.

The compound that is used to elicit an immunogenic response is referredto as an immunogen or antigen. An “epitope” or “antigenic determinant”is an area on the surface of an immunogen that stimulates a specificimmune response directed to the epitope. In proteins, particularlydenatured proteins, an epitope is typically defined and represented by acontiguous amino acid sequence. However, in the case of nondenaturedproteins, epitopes also include structures, such as active sites, thatare formed by the three-dimensional folding of a protein in a mannersuch that amino acids from separate portions of the amino acid sequenceof the protein are brought into close physical contact with each other.

A “hapten” is a small molecule that cannot provoke an immune responseunless first bound to an immunogenic carrier molecule. Although a haptencannot itself provoke an immune response, it is specifically bound byantibodies generated during an immunogenic response to thehapten-carrier conjugate.

The term “antibody fragment” refers to functional fragments ofantibodies, i.e., polypeptides that are smaller than an antibody whichhave sequences from the antibody, but nevertheless have the ability tospecifically bind to an antigenic determinant. Antibody fragments can beprepared by in vitro manipulation of antibodies (e.g., by limitedproteolysis of an antibody), or via recombinant DNA technology (e.g.,the preparation of single-chain antibodies from phage displaylibraries).

XI.B. Antibody Structure

Naturally occurring (wildtype) antibody molecules are Y-shaped moleculesconsisting of four polypeptide chains, two identical heavy chains andtwo identical light chains, which are covalently linked together bydisulfide bonds. Both types of polypeptide chains have constant regions,which do not vary or vary minimally among antibodies of the same class(i.e., IgA, IgM, etc.), and variable regions. The variable regions areunique to a particular antibody and comprise a recognition element foran epitope. The carboxy-terminal regions of both heavy and light chainsare conserved in sequence and are called the constant regions (alsoknown as C-domains). The amino-terminal regions (also known asV-domains) are variable in sequence and are responsible for antibodyspecificity. The antibody specifically recognizes and binds to anantigen mainly through six short complementarity-determining regions(CDRs) located in their V-domains.

Each light chain of an antibody is associated with one heavy chain, andthe two chains are linked by a disulfide bridge formed between cysteineresidues in the carboxy-terminal region of each chain, which is distalfrom the amino terminal region of each chain that constitutes itsportion of the antigen binding domain. Antibody molecules are furtherstabilized by disulfide bridges between the two heavy chains in an areaknown as the hinge region, at locations nearer the carboxy terminus ofthe heavy chains than the locations where the disulfide bridges betweenthe heavy and light chains are made. The hinge region also providesflexibility for the antigen-binding portions of an antibody.

An antibody's specificity is determined by the variable regions locatedin the amino terminal regions of the light and heavy chains. Thevariable regions of a light chain and associated heavy chain form an“antigen binding domain” that recognizes a specific epitope; an antibodythus has two antigen binding domains. The antigen binding domains in awildtype antibody are directed to the same epitope of an immunogenicprotein, and a single wildtype antibody is thus capable of binding twomolecules of the immunogenic protein at the same time. Thus, a wildtypeantibody is monospecific (i.e., directed to a unique antigen) anddivalent (i.e., capable of binding two molecules of antigen).

XI.C. Types of Antibodies

“Polyclonal antibodies” are generated in an immunogenic response to aprotein having many epitopes. A composition (e.g., serum) of polyclonalantibodies thus includes a variety of different antibodies directed tothe same and to different epitopes within the protein. Methods forproducing polyclonal antibodies are known in the art (see, e.g., Cooperet al., Section III of Chapter 11 in: Short Protocols in MolecularBiology, 2nd Ed., Ausubel et al., eds., John Wiley and Sons, New York,1992, pages 11-37 to 11-41).

“Antipeptide antibodies” (also known as “monospecific antibodies”) aregenerated in a humoral response to a short (typically, 5 to 20 aminoacids) immunogenic polypeptide that corresponds to a few (preferablyone) isolated epitopes of the protein from which it is derived. Aplurality of antipeptide antibodies includes a variety of differentantibodies directed to a specific portion of the protein, i.e, to anamino acid sequence that contains at least one, preferably only one,epitope. Methods for producing antipeptide antibodies are known in theart (see, e.g., Cooper et al., Section III of Chapter 11 in: ShortProtocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., JohnWiley and Sons, New York, 1992, pages 11-42 to 11-46).

A “monoclonal antibody” is a specific antibody that recognizes a singlespecific epitope of an immunogenic protein. In a plurality of amonoclonal antibody, each antibody molecule is identical to the othersin the plurality. In order to isolate a monoclonal antibody, a clonalcell line that expresses, displays and/or secretes a particularmonoclonal antibody is first identified; this clonal cell line can beused in one method of producing the antibodies of the invention. Methodsfor the preparation of clonal cell lines and of monoclonal antibodiesexpressed thereby are known in the art (see, for example, Fuller et al.,Section II of Chapter 11 in: Short Protocols in Molecular Biology, 2ndEd., Ausubel et al., eds., John Wiley and Sons, New York, 1992, pages11-22 to 11-11-36).

A “naked antibody” is an antibody that lacks the Fc portion of awildtype antibody molecule. The Fc portion of the antibody moleculeprovides effector functions, such as complement fixation and ADCC(antibody dependent cell cytotoxicity), which set mechanisms into actionthat may result in cell lysis. See, e.g., Markrides, Therapeuticinhibition of the complement system, Pharmacol. Rev. 50:59-87, 1998. Insome systems, it appears that the therapeutic action of an antibodydepends upon the effector functions of the Fc region (see, e.g., Golayet al., Biologic response of B lymphoma cells to anti-CD20 monoclonalantibody rituximab in vitro: CD55 and CD59 regulate complement-mediatedcell lysis, Blood 95:3900-3908, 2000).

However, it is possible that the Fc portion is not required fortherapeutic function in every instance, as other mechanisms, such asapoptosis, can come into play. Moreover, the Fc region may bedeleterious in some applications as antibodies comprising an Fc regionare taken up by Fc receptor-bearing cells, thereby reducing the amountof thereapeutic antibody taken up targeted cells. Vaswani and Hamilton,Humanized antibodies as potential therapeutic drugs. Ann. Allergy AsthmaImmunol. 81:105-119, 1998. Components of the immune system may recognizeand react to antibodies that are clumped together on the surface oftumor cells. It is thus envisioned that the resulting immune responsewill target and destroy, or at least limit the proliferation of, thetumor cells.

One way to get naked antibodies deilivered to surfaces where they willclump together is to use a targetable construct or complex to bringdifferent naked antibodies together on a targeted cellular surface. Byway of non-limiting example, an anti-C20 antibody (e.g., Rituxan) and ananti-C22 antibody might be administered separately or together, allowedto clear so that unbound antibodies are removed from the system. Theaddition of a targetable construct that binds and connects both typesantibodies, thereby forming a targetable construct in situ, which isexpected to mimic a group of anti-C20 and anti-C22 antibodies clumped onthe surface of a tumor cell.

Naked antibodies are also of interest for therapy of diseases caused byparasites, such as malaria. Vukovic et al., Immunoglobulin G3 antibodiesspecific for the 19-kilodalton carboxyl-terminal fragment of Plasmodiumyoelii merozoite surface protein 1 transfer protection to mice deficientin Fc-

RI receptors, Infect. Immun. 68:3019-22, 2000.

Single chain antibodies (scFv) generally do not include portions of theFc region of antibodies that are involved in effector functions and arethus naked antibodies, although methods are known for adding suchregions to known scFv molecules if desired. See Helfrich et al., A rapidand versitile method for harnessing scFv antibody fragments with variousbiological functions, J. Immunol. Meth. 237:131-145,2000; and de Haardet al., Creating and engineering human antibodies for immunotherapy,Adv. Drug Delivery Rev. 31:5-31, 1998.

XI.D. Antibody Fragments

XI.D. 1. Proteolytic Antibody Fragments

Antibody fragments produced by limited proteolysis of wildtypeantibodies are called proteolytic antibody fragments. These include, butare not limited to, the following.

“F(ab′)₂ fragments” are released from an antibody by limited exposure ofthe antibody to a proteolytic enzyme, e.g., pepsin or ficin. An F(ab′)₂fragment comprises two “arms,” each of which comprises a variable regionthat is directed to and specifically binds a common antigen. The twoFab′ molecules are joined by interchain disulfide bonds in the hingeregions of the heavy chains; the Fab′ molecules may be directed towardthe same (bivalent) or different (bispecific) epitopes.

“Fab′ fragments” contain a single anti-binding domain comprising an Faband an additional portion of the heavy chain through the hinge region.

“Fab′-SH fragments” are typically produced from F(ab′)₂ fragments, whichare held together by disulfide bond(s) between the H chains in anF(ab′)₂ fragment. Treatment with a mild reducing agent such as, by wayof non-limiting example, beta-mercaptoethylamine, breaks the disulfidebond(s), and two Fab′ fragments are released from one F(ab′)₂ fragment.Fab′-SH fragments are monovalent and monospecific.

“Fab fragments” (i.e., an antibody fragment that contains theantigen-binding domain and comprises a light chain and part of a heavychain bridged by a disulfide bond) are produced by papain digestion ofintact antibodies. A convenient method is to use papain immobilized on aresin so that the enzyme can be easily removed and the digestionterminated. Fab fragments do not have the disulfide bond(s) between theH chains present in an F(ab′)₂ fragment.

XI.D.2. Recombinant Antibody Fragments

“Single-chain antibodies” are one type of antibody fragment. The termsingle chain antibody is often abbreviated as “scFv” or “sFv.” Theseantibody fragments are produced using molecular genetics and recombinantDNA technology. A single-chain antibody consists of a polypeptide chainthat comprises both a V_(H) and a V_(L) portion. Unlike wildtypeantibodies, wherein two separate heavy and light polypeptide chains areconjoined to form a single antigen-binding variable region, asingle-chain antibody is a single polypeptide that comprises anantigen-binding variable region. That is, a single-chain antibodycomprises the variable, antigen-binding determinative region of a singlelight and heavy chain of an antibody linked together by a chain of 10-25amino acids.

The term “single-chain antibody” includes but is not limited to adisulfide-linked Fv (dsFv) in which two single-chain antibodies linkedtogether by a disulfide bond; a bispecific sFv (a sFv or a dsFv moleculehaving two antigen-binding domains, each of which may be directed to adifferent epitope); a diabody (a dimerized sFv formed when the V_(H)domain of a first sFv assembles with the V_(L) domain of a second sFvand the V_(L) domain of the first sFv assembles with the V_(H) domain ofthe second sFv; the two antigen-binding regions of the diabody may bedirected towards the same or different epitopes); and a triabody (atrimerized sFv, formed in a manner similar to a diabody, but in whichthree antigen-binding domains are created in a single complex; the threeantigen binding domains may be directed towards the same or differentepitopes).

“Camelid antibodies” are unlike mammalian antibodies in that they needonly V-domain, namely V_(H), to specifically and effectively bind anantigen. Camelid antibodies or fragments thereof have the advantages ofbeing water soluble and showing good expression in yeast and Aspergillusmoulds. For reviews, see Muyldermans, Single domain camel antibodies:current status, J. Biotechnol. 74:277-302, 2001; and Wemery, Camelidimmunoglobulins and their importance for the new-born—a review, J. Vet.Med. B. Infect. Dis. Vet. Public Health 48:561-8, 2001. See alsoSpinelli et al., Camelid heavy-chain variable domains provide efficientcombining sites to haptens, Biochemistry 39:1217-22, 2000; Muyldermanset al., Unique single-domain antigen binding fragments derived fromnaturally occurring camel heavy-chain antibodies, J. Mol. Recog.12:131-40, 1999; and Davies et al., Single antibody domains as smallrecognition units: design and in vitro antigen selection of camelized,human V_(H) domains with improved protein stability, Protein Eng.9:531-7, 1996. Other immunoglobulin-like molecules from other speciesmay also be used. See, e.g., Roux et al., Structural analysis of thenurse shark (new) antigen receptor (NAR): molecular convergence of NARand unusual mammalian immunoglobulins, Proc. Natl. Acad. Sci. USA.95:11804-9, 1998. Methods of producing camelid antibodies are known inthe art. See, for example, U.S. Pat. Nos. 6,015,695; 6,005,079;5,874,541; 5,840,526; 5,800,988; and 5,759,808, each of which isentitled Immunoglobulins Devoid of Light Chains.

“Humanized antibodies” have been modified, by genetic manipulationand/or in vitro treatment to be more human, in terms of amino acidsequence, glycosylation pattern, etc., in order to reduce theantigenicity of the antibody or antibody fragment in an animal to whichthe antibody is intended to be administered. See Gussow and Seemann,Humanization of monoclonal antibodies, Meth. Enz. 203:99-121, 1991 andVaswani and Hamilton, Humanized antibodies as potential therapeuticdrugs, Ann. Allergy Asthma Immunol. 81:105-119, 1998.

“Fully human antibodies” are human antibodies produced in transgenicanimals such as Xenomice. XenoMouse strains are genetically engineeredmice in which the murine IgH and Igk loci have been functionallyreplaced by their Ig counterparts on yeast artificial YAC transgenes.These human Ig transgenes can carry the majority of the human variablerepertoire and can undergo class switching from IgM to IgG isotypes. Theimmune system of the xenomouse recognizes administered human antigens asforeign and produces a strong humoral response. The use of XenoMouse inconjunction with well-established hybridomas techniques, results infully human IgG mAbs with sub-nanomolar affinities for human antigens(see U.S. Pat. No. 5,770,429, entitled “Transgenic non-human animalscapable of producing heterologous antibodies”, U.S. Pat. No. 6,162,963,entitled “Generation of xenogenetic antibodies”; U.S. Pat. No.6,150,584, entitled “Human antibodies derived from immunized xenomice”,U.S. Pat. No. 6,114,598, entitled “Generation of xenogeneic antibodies”;and U.S. Pat. No. 6,075,181, entitled “Human antibodies derived fromimmunized xenomice”; for reviews, see Green, Antibody engineering viagenetic engineering of the mouse: XenoMouse strains are a vehicle forthe facile generation of therapeutic human monoclonal antibodies, J.Immunol. Meth. 231:11-23, 1999; Wells, Eek, a XenoMouse: Abgenix, Inc.,Chem. Biol. 7:R185-6, 2000; and Davis et al., Transgenic mice as asource of filly human antibodies for the treatment of cancer, CancerMetastasis Rev. 18:421-5, 1999).

“Complementary determining region peptides” or “CDR peptides” areanother form of an antibody fragment. A CDR peptide (also known as“minimal recognition unit”) is a peptide corresponding to a singlecomplementarity-determining region (CDR), and can be prepared byconstructing genes encoding the CDR of an antibody of interest. Suchgenes are prepared, for example, by using the polymerase chain reactionto synthesize the variable region from RNA of antibody-producing cells.See, for example, Larrick et al., Methods: A Companion to Methods inEnzymology 2:106, 1991.

“T-cell receptor (TCR) fragments” are soluble peptides having amino acidsequences corresponding to the variable and constant regions of a T-cellreceptor. Soluble TCR fragments can be prepared as a single chain(scTCR) or as separate components with dimerization domain that allowthe separate peptides to stably associated with each other. (Willcox etal., Production of soluble alpha:beta T-cell receptor heterodimerssuitable for biophysical analysis of ligand binding, Protein Science8:2418-2423, 1999). Soluble TCR molecules made using chinese hamsterovary (CHO) cells are described by Lin et al., Expression of T CellAntigen Receptor Heterodimers in a Lipid-Linked Form, Science249:677-679, 1990; and Davis et al., TCR Recognition and Selection InVivo, Cold Spring Harbor Symposia on Quantitative Biology, LIV, 119-128,1989). Both of these articles describe the use of a GPI linkage approachto produce soluble TCR molecules. For other examply methods of producingsoluble TCR fragments, see, for example, U.S. Pat. No. 6,165,745,Recombinant production of immunoglobulin-like domains in prokaryoticcells; U.S. Pat. No. 6,080,840, Soluble T cell receptors; U.S. Pat. No.5,723,309, Production of subunits of soluble T cell receptors byco-transfection; U.S. Pat. No. 5,552,300, T cell antigen receptor Vregion proteins and methods of preparation thereof; Novotny et al.,Proc. Natl. Acad Sci USA. 88:8646-8650, 1991; Ward, Scand. J. Immunol.34:215-220, 1991; and Pecorari et al., Folding, HeterodimericAssociation and Specific Peptide Recognition of a Murine αβ T-cellReceptor Expressed in Escherichia coli, J. Mol. Biol. 285:1831-1843,1999.

“Chimeric antibody derivatives” such as chimeric TCR:Ab molecules havebeen produced by shuffling the variable and constant domains of murineT-cell receptors with the constant region of an immunoglobulin kappalight chain. For example, Gregoire et al. (Proc. Natl. Acad. Sci. USA88:8077-8081, 1991) show a murine chimera consisting of the C-alpha andV-alpha genes of the KB5-C2 joined to the C region of the kappa lightchain of the S105 monoclonal antibody, and a V-beta-C-beta-C-kappa.chimera. Both are transfected into a mammalian B cell myeloma that doesnot express native immunoglobulin heavy or light chains. See also, Weberet al., Nature 356:793-795, 1992.

In “cysteine-modified antibodies,” a cysteine amino acid is inserted orsubstituted on the surface of antibody by genetic manipulation and usedto conjugate the antibody to another molecule via, e.g., a disulfidebridge. Cysteine substitutions or insertions for antibodies have beendescribed (see U.S. Pat. No. 5,219,996). Methods for introducing Cysresidues into the constant region of the IgG antibodies for use insite-specific conjugation of antibodies are described by Stimmel et al.(J. Biol. Chem 275:330445-30450, 2000).

XII. Pharmaceutical Compositions

XII.A. Definitions

A “pharmaceutical composition” refers to a composition comprising a drugwherein the carrier is a pharmaceutically acceptable carrier, while a“veterinary composition” is one wherein the carrier is a veterinarilyacceptable carrier. The term “pharmaceutically acceptable carrier” or“veterinarily acceptable carrier” includes any medium or material thatis not biologically or otherwise undesirable, i.e, the carrier may beadministered to an organism along with a composition or compound of theinvention without causing any undesirable biological effects orinteracting in a deleterious manner with the complex or any of itscomponents or the organism. Examples of pharmaceutically acceptablereagents are provided in The United States Pharmacopeia, The NationalFormulary, United States Pharmacopeial Convention, Inc., Rockville, Md.1990, hereby incorporated in its entirety by reference herein into thepresent application, as is Phamaceutical Dosage Forms & Drug DeliverySystems, 7th Edition, Ansel et al., editors, Lippincott Williams &Wilkins, 1999.

The drug (i.e., targetable construct or complex) is included in thepharmaceutical composition in an amount sufficient to produce thedesired effect upon the patient. The pharmaceutical compositions of theinvention can further comprise other chemical components, such asdiluents and excipients. A “diluent” is a chemical compound diluted in asolvent, preferably an aqueous solvent, that facilitates dissolution ofthe drug in the solvent, and it may also serve to stabilize thebiologically active form of the drug or one or more of its components.Salts dissolved in buffered solutions are utilized as diluents in theart. For example, preferred diluents are buffered solutions containingone or more different salts. A preferred buffered solution is phosphatebuffered saline (particularly in conjunction with compositions intendedfor pharmaceutical administration), as it mimics the salt conditions ofhuman blood. Since buffer salts can control the pH of a solution at lowconcentrations, a buffered diluent rarely modifies the biologicalactivity of a biologically active peptide.

An “excipient” is any more or less inert substance that can be added toa composition in order to confer a suitable property, for example, asuitable consistency or to form a drug. Suitable excipients and carriersinclude, in particular, fillers such as sugars, including lactose,sucrose, mannitol, or sorbitol cellulose preparations such as, forexample, maize starch, wheat starch, rice starch, agar, pectin, xanthangum, guar gum, locust bean gum, hyaluronic acid, casein potato starch,gelatin, gum tragacanth, polyacrylate, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents can alsobe included, such as cross-linked polyvinylpyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Other suitableexcipients and carriers include hydrogels, gellable hydrocolloids, andchitosan. Chitosan microspheres and microcapsules can be used ascarriers. See WO 98/52547 (which describes microsphere formulations fortargeting compounds to the stomach, the formulations comprising an innercore (optionally including a gelled hydrocolloid) containing one or moreactive ingredients, a membrane comprised of a water insoluble polymer(e.g., ethylcellulose) to control the release rate of the activeingredient(s), and an outer layer comprised of a bioadhesive cationicpolymer, for example, a cationic polysaccharide, a cationic protein,and/or a synthetic cationic polymer; U.S. Pat. No. 4,895,724. Typically,chitosan is cross-linked using a suitable agent, for example,glutaraldehyde, glyoxal, epichlorohydrin, and succinaldehyde.Compositions employing chitosan as a carrier can be formulated into avariety of dosage forms, including pills, tablets, microparticles, andmicrospheres, including those providing for controlled release of theactive ingredient(s). Other suitable bioadhesive cationic polymersinclude acidic gelatin, polygalactosamine, polyamino acids such aspolylysine, polyhistidine, polyornithine, polyquaternary compounds,prolamine, polyimine, diethylaminoethyldextran (DEAE), DEAE-imine,DEAE-methacrylate, DEAE-acrylamide, DEAE-dextran, DEAE-cellulose,poly-p-aminostyrene, polyoxethane, copolymethacrylates, polyamidoamines,cationic starches, polythiodiethylaminomethylethylene andpolyvinylpyridine.

XII.B. Formulation of Pharmaceutical Compositions

The targetable constructs and complexes of the invention can beformulated in any suitable manner. The targetable constructs andcomplexes may be uniformly (homogeneously) or non-uniformly(heterogenously) dispersed in the carrier. Suitable formulations includedry and liquid formulations. Dry formulations include freeze dried andlyophilized powders, which are particularly well suited for aerosoldelivery to the sinuses or lung, or for long term storage followed byreconstitution in a suitable diluent prior to administration. Otherpreferred dry formulations include those wherein a pharmaceuticalcomposition according to the invention is compressed into tablet or pillform suitable for oral administration or compounded into a sustainedrelease formulation. When the pharmaceutical composition is intended fororal administration but the targetable construct or complex is to bedelivered to epithelium in the intestines, it is preferred that theformulation be encapsulated with an enteric coating to protect theformulation and prevent premature release of the targetable constructsand complexes included therein. As those in the art will appreciate, thepharmaceutical compositions of the invention can be placed into anysuitable dosage form. Pills and tablets represent some of such dosageforms. The pharmaceutical compositions can also be encapsulated into anysuitable capsule or other coating material, for example, by compression,dipping, pan coating, spray drying, etc. Suitable capsules include thosemade from gelatin and starch. In turn, such capsules can be coated withone or more additional materials, for example, and enteric coating, ifdesired. Liquid formulations include aqueous formulations, gels, andemulsions.

Some preferred embodiments concern compositions that comprise abioadhesive, preferably a mucoadhesive, coating. A “bioadhesive coating”is a coating that allows a drug to adhere to a biological surface orsubstance better than occurs absent the coating. A “mucoadhesivecoating” is a preferred bioadhesive coating that allows a substance, forexample, a composition according to the invention, to adhere better tomucosa occurs absent the coating. For example, micronized particles(e.g., particles having a mean diameter of about 5, 10, 25, 50, or 100

m) can be coated with a mucoadhesive. The coated particles can then beassembled into a dosage form suitable for delivery to an organism.Preferably, and depending upon the location where the cell surfacetransport moiety to be targeted is expressed, the dosage form is thencoated with another coating to protect the formulation until it reachesthe desired location, where the mucoadhesive enables the formulation tobe retained while the compositions or compounds of the inventioninteract with the target cell surface transport moiety.

XII.C. Administration of Pharmaceutical Compositions

The pharmaceutical compositions of the invention facilitateadministration of monoclonal antibodies to an organism, preferably ananimal, preferably a mammal, bird, fish, insect, or arachnid. Preferredmammals include bovine, canine, equine, feline, ovine, and porcineanimals, and non-human primates. Humans are particularly preferred.Multiple techniques of administering or delivering a compound exist inthe art including, but not limited to, oral, rectal (e.g., an enema orsuppository) aerosol (e.g., for nasal or pulmonary delivery),parenteral, and topical administration. Preferably, sufficientquantities of the composition or compound of the invention are deliveredto achieve the intended effect. The particular amount of composition orcompound to be delivered will depend on many factors, including theeffect to be achieved, the type of organism to which the composition isdelivered, delivery route, dosage regimen, and the age, health, and sexof the organism. As such, the particular dosage of a composition orcompound of the invention included in a given formulation is left to theordinarily skilled artisan's discretion.

Those skilled in the art will appreciate that when the pharmaceuticalcompositions of the present invention are administered as agents toachieve a particular desired biological result, which may include atherapeutic or protective effect(s) (including vaccination), it may benecessary to combine the composition or compound of the invention with asuitable pharmaceutical carrier. The choice of pharmaceutical carrierand the preparation of the composition or compound as a therapeutic orprotective agent will depend on the intended use and mode ofadministration. Suitable formulations and methods of administration oftherapeutic agents include, but are not limited to, those for oral,pulmonary, nasal, buccal, ocular, dermal, rectal, or vaginal delivery.

Depending on the mode of delivery employed, the context-dependentfunctional entity can be delivered in a variety of pharmaceuticallyacceptable forms. For example, the context-dependent functional entitycan be delivered in the form of a solid, solution, emulsion, dispersion,micelle, liposome, and the like, incorporated into a pill, capsule,tablet, suppository, areosol, droplet, or spray. Pills, tablets,suppositories, areosols, powders, droplets, and sprays may have complex,multilayer structures and have a large range of sizes. Aerosols,powders, droplets, and sprays may range from small (1 micron) to large(200 micron) in size.

Pharmaceutical compositions of the present invention can be used in theform of a solid, a lyophilized powder, a solution, an emulsion, adispersion, a micelle, a liposome, and the like, wherein the resultingcomposition contains one or more of the targetable constructs orcomplexes of the present invention, as an active ingredient, inadmixture with an organic or inorganic carrier or excipient suitable forenteral or parenteral applications. The active ingredient may becompounded, for example, with the usual non-toxic, pharmaceuticallyacceptable carriers for tablets, pellets, capsules, suppositories,solutions, emulsions, suspensions, and any other form suitable for use.The carriers which can be used include glucose, lactose, mannose, gumacacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc,corn starch, keratin, colloidal silica, potato starch, urea, mediumchain length triglycerides, dextrans, and other carriers suitable foruse in manufacturing preparations, in solid, semisolid, or liquid form.In addition auxiliary, stabilizing, thickening and coloring agents andperfumes may be used. Examples of a stabilizing dry agent includestriulose, preferably at concentrations of 0.1% or greater (See, e.g.,U.S. Pat. No. 5,314,695).

XII.D. Dosages

Although individual needs may vary, determination of optimal ranges foreffective amounts of pharmaceutical compositions is within the skill ofthe alt. Human doses can be extrapolated from animal studies (Katocs etal., Chapter 27 In: Remington's Pharmaceutical Sciences, 18th Ed.,Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990). Generally, thedosage required to provide an effective amount of a pharmaceuticalcomposition, which can be adjusted by one skilled in the art, will varydepending on the age, health, physical condition, weight, type andextent of the disease or disorder of the recipient, frequency oftreatment, the nature of concurrent therapy (if any) and the nature andscope of the desired effect(s). See, for example, Nies et al., Chapter 3In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9thEd., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996)

Dosing of therapeutic compositions is dependent on severity andresponsiveness of the disease state to be treated, with the course oftreatment lasting from several days to several months, or until a cureis effected or a diminution of the disease state is achieved. Optimaldosing schedules can be calculated from measurements of drugaccumulation in the body of the patient. The term “patient” is intendedto encompass animals (e.g., cats, dogs and horses) as well as humans.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual therapeutic agents, and cangenerally be estimated based on EC₅₀ found to be effective in in vitroand in vivo animal models.

The range of doses (the amount of targetable construct or complexadministered) is broad, since in general the efficacy of a therapeuticeffect for different mammals varies widely with doses typically being20, 30 or even 40 times smaller (per unit body weight) in man than inthe rat. In general, dosage is from 0.01

g to 100 g per kg of body weight, preferably 0.01

g to 10 g/kg of body weight, 0.01

g to 1000 mg/kg of body weight, 0.01

g to 100 mg/kg of body weight, 0.01

g to 10 mg/kg of body weight, 0.01

g to 1 mg/kg of body weight, 0.01

g to to 100

g/kg of body weight, 0.01

g to to 10

g/kg of body weight, 0.01

g to 1

g/kg of body weight, 0.01

g to 10

g/kg of body weight, 0.01

g to 1

g/kg of body weight, 0.01

g to 0.1

g/kg of body weight, and ranges based on the boundaries of the precedingranges of concentrations. Thus, for example, the preceding descriptionof dosages encompasses dosages within the range of 100 to 10 g per kg ofbody weight, 10 g to 1000 mg/kg of body weight, 1000 mg to 100 mg, etc.

Doses may be given once or more daily, weekly, monthly or yearly, oreven once every 2 to 20 years. Persons of ordinary skill in the art caneasily estimate repetition rates for dosing based on measured residencetimes and concentrations of the targetable construct or complex inbodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the therapeutic agent isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

The specific dose is calculated according to the approximate body weightor surface area of the patient. Other factors in determining theappropriate dosage can include the disease or condition to be treated orprevented, the severity of the disease, the route of administration, andthe age, sex and medical condition of the patient. Further refinement ofthe calculations necessary to determine the appropriate dosage fortreatment is routinely made by those skilled in the art, especially inlight of the dosage information and assays disclosed herein. The dosagecan also be determined through the use of known assays for determiningdosages used in conjunction with appropriate dose-response data.

An individual patient's dosage can be adjusted as the progress of thedisease is monitored. Blood levels of the targetable construct orcomplex in a patient can be measured to see if the dosage needs to beadjusted to reach or maintain an effective concentration.Pharmacogenomics may be used to determine which targetable constructsand/or complexes, and dosages thereof, are most likely to be effectivefor a given individual (Schmitz et al., Clinica Chimica Acta 308:43-53,2001; Steimer et al., Clinica Chimica Acta 308:33-41, 2001).

XIII. References, Patents and Published Patent Applications

-   -   XIII.A. Scientific References

Bamias, A., and Epenetos, A. A. Two-step strategies for the diagnosisand treatment of cancer with bioconjugates. Antibody, Immunoconjugates,Radiopharm. 1992; 5: 385-395.

Barbet, J., Peltier, P., Bardet, S., Vuillez, JP., Bachelot, I., Denet,S., Olivier, P., Lecia, F., Corcuff, B., Huglo, D., Proye, C., Rouvier,E., Meyer, P., Chatal, J. F. Radioimmunodetection of medullary thyroidcarcinoma using indium-111 bivalent hapten and anti-CEA xanti-DTPA-indium bispecifc antibody. J. Nucl. Med. 1998; 39:1172-1178.

Bos, ES., Kuijpers, W H A., Meesters-Winters, M., Pham, D T., deHaan, AS., van Doormalen, Am., Kasperson, F. M., vanBoeckel, C A A andGouegeon-Bertrand, F. In vivo evaluation of DNA-DNA hybridization as atwo-step approach in radioimmunotherapy of cancer. Cancer Res. 1994;54:3479-3486.

Gautherot, E., Bouhou, J., LeDoussal, J-M., Manetti, C., Martin, M.,Rouvier, E., Barbet, J. Therapy for colon carcinoma xenografts withbi-specific antibody-targeted, iodine-131-labeled bivalent hapten.Cancer suppl. 1997; 80: 2618-2623.

Gautherot, E., Bouhou, J., Loucif, E., Manetti, C., Martin, M.,LeDoussal, J. M., Rouvier, E., Barbet, J. Radioimmunotherapy of LS 174Tcolon carcinoma in nude mice using an iodine-131-labeled bivalent haptencombined with an anti-CEA x anti-indium-DTPA bi-specific antibody. J.Nucl. Med. Suppl. 1997; 38: 7p.

Goodwin, D. A., Meares, CF., McCall, M J., McTigue, M., Chaovapong, W.Pre-targeted immunoscintigraphy of murine tumors with indium-111-labeledbifunctional haptens. J.Nucl.Med. 1988; 29:226-234.

Greenwood, F. C. and Hunter, W. M. The preparation of 1-131 labeledhuman growth hormone of high specific radioactivity. Biochem. 1963;89:114-123.

Hawkins, G. A., McCabe, R. P., Kim, C.-H., Subramanian,R., Bredehorst,R., McCullers, G. A., Vogel, C.-W., Hanna, M. G. Jr., and Pomata, N.Delivery of radionuclides to pretargeted monoclonal antibodies usingdihydrofolate reductase and methotrexate in an affinity system. CancerRes. 1993; 53: 2368-2373.

Kranenborg, M. h., Boerman, O. C., Oosterwijk-Wakka, j., weijert, M.,Corstens, F., Oosterwijk, E. Development and characterization ofanti-renal cell carcinoma x antichelate bi-specific monoclonalantibodies for two-phase targeting of renal cell carcinoma. CancerRes.(suppl) 1995; 55: 5864s-5867s.

Penefsky, H. S. A centrifuged column procedure for the measurement ofligand binding by beef heart F1. Part G. Methods Enzymol. 1979;56:527-530.

Schuhmacher, J., Klivenyi,G., Matys, R., Stadler, M., Regiert, T.,Hauser, H., Doll, J., Maier-Borst, W., Zoller, M. Multistep tumortargeting in nude mice using bi-specific antibodies and a galliumchelate suitable for immunocintigraphy with positron emissiontomography. Cancer Res. 1995; 55, 115-123.

Sharkey, R M., Karacay, H. Griffiths, G L., Behr, T M., Blumenthal, RD., Mattes, M J., Hansen, H J., Goldenberg, D M. Development of astreptavidin-anti-carcinoembryonic antigen antibody, radiolabeled biotinpretargeting method for radioimmunotherapy of colorectal cancer. Studiesin a human colon cancer xenograft model. Bioconjugate Chem 1997;8:595-604.

Stickney, D R., Anderson, L D., Slater, J B., Ahlem, C N., Kirk, G A.,Schweighardt, S A and Frincke, J M. Bifunctional antibody: a binaryradiopharmaceutical delivery system for imaging colorectal carcinoma.Cancer Res. 1991;51: 6650-6655.

XIII.B. U.S. Patents

U.S. Pat. No. 6,358,489, Fluorination of proteins and peptides for F-18positron emission tomography

U.S. Pat. No. 6,331,175, Method and kit for imaging and treating organsand tissues.

U.S. Pat. No. 6,319,500, Detection and treatment of infections withimmunoconjugates.

U.S. Pat. No. 6,306,393, Immunotherapy of B-cell malignancies usinganti-CD22 antibodies.

U.S. Pat. No. 6,254,868, Glycosylated humanized B-cell specificantibodies.

U.S. Pat. No. 6,228,362, Boron neutron capture therapy usingpre-targeting methods.

U.S. Pat. No. 6,187,287, Immunoconjugates and humanized antibodiesspecific for B-cell lymphoma and leukemia cells.

U.S. Pat. No. 6,187,284, Fluorination of proteins and peptides for F-18positron emission tomography.

U.S. Pat. No. 6,183,744, Immunotherapy of B-cell malignancies usinganti-CD22 antibodies.

U.S. Pat. No. 6,132,718, Multi-stage cascade boosting vaccine.

U.S. Pat. No. 6,126,916, Radiometal-binding peptide analogues.

U.S. Pat. No. 6,120,768, Dota-biotin derivatives.

U.S. Pat. No. 6,096,289, Intraoperative, intravascular, and endoscopictumor and lesion detection, biopsy and therapy.

U.S. Pat. No. 6,090,381 Stimulation of an immune response withantibodies labeled with the α-galactosyl epitope.

U.S. Pat. No. 6,083,477 Non-antigenic toxin-conjugate and fusion proteinof internalizing receptor system.

U.S. Pat. No. 6,077,499 Targeted combination immunotherapy of cancer

U.S. Pat. No. 6,071,490 Position emission tomography using gallium-68chelates

U.S. Pat. No. 6,010,680 Thiolation of proteins for radionuclide-basedradioimmunodetection and radioimmunotherapy

U.S. Pat. No. 5,976,492 Radioactive phosphorus labeled proteins fortargeted radiotherapy

U.S. Pat. No. 5,965,131 Delivery of diagnostic and therapeutic agents toa target site

U.S. Pat. No. 5,958,408 Delivery of diagnostic and therapeutic agents toa target site

U.S. Pat. No. 5,922,302 Detection and therapy of lesions withbiotin/avidin-metal chelating protein conjugates

U.S. Pat. No. 5,874,540 CDR-grafted type III anti-CEA humanized mousemonoclonal antibodies

U.S. Pat. No. 5,851,527 Method for antibody targeting of therapeuticagents

U.S. Pat. No. 5,846,741 Boron neutron capture therapy usingpre-targeting methods

U.S. Pat. No. 5,843,397 Cytotoxic therapy for graft rejection

U.S. Pat. No. 5,798,100 Multi-stage cascade boosting vaccine

U.S. Pat. No. 5,789,554 Immunoconjugates and humanized antibodiesspecific for B-cell lymphoma and leukemia cells

U.S. Pat. No. 5,776,095 Method and kit for imaging and treating organsand tissues

U.S. Pat. No. 5,776,094 Method and kit for imaging and treating organsand tissues

U.S. Pat. No. 5,776,093 Method for imaging and treating organs andtissues

U.S. Pat. No. 5,772,981 Thiolation of proteins for radionuclide-basedradioimmunodetection and radioimmunotherapy

U.S. Pat. No. 5,753,206 Radiometal-binding analogues of luteinizinghormone releasing hormone

U.S. Pat. No. 5,746,996 Thiolation of peptides for radionuclide-basedradiodetection and radiotherapy

U.S. Pat. No. 5,736,119 Detection and therapy of lesions withbiotin/avidin-metal chelating protein conjugates

U.S. Pat. No. 5,728,369 Radioactive phosphorus labeling of proteins fortargeted radiotherapy

U.S. Pat. No. 5,716,595 Intraoperative, intravascular and endoscopictumor and lesion detection and therapy with monovalent antibodyfragments

U.S. Pat. No. 5,705,158 Treatment of infectious and inflammatory lesions

U.S. Pat. No. 5,698,405 Method of reducing immunogenicity

U.S. Pat. No. 5,698,178 Polyspecific immunoconjugates and antibodycomposites for targeting the multidrug resistant phenotype

U.S. Pat. No. 5,697,902 Method for imaging and treating organs andtissues

U.S. Pat. No. 5,686,578 Polyspecific immunoconjugates and antibodycomposites for targeting the multidrug resistant phenotype

U.S. Pat. No. 5,677,427 Chimeric antibody for detection and therapy ofinfectious and inflammatory lesions

U.S. Pat. No. 5,670,132 Modified radioantibody fragments for reducedrenal uptake

U.S. Pat. No. 5,637,288 Chimeric antibody for detection and therapy ofinfectious and inflammatory lesions

U.S. Pat. No. 5,635,603 Preparation and use of immunoconjugates

U.S. Pat. No. 5,632,968 Detection of cardiovascular lesions

U.S. Pat. No. 5,612,016 Conjugates of antibodies and bifunctionalligands

U.S. Pat. No. 5,609,846 Radiolabelled antibody cytotoxic therapy ofinfectious or autoimmune disease

U.S. Pat. No. 5,601,825 Therapeutic conjugates of toxins and drugs

U.S. Pat. No. 5,541,297 Therapeutic conjugates of toxins and drugs

U.S. Pat. No. 5,525,338 Detection and therapy of lesions withbiotin/avidin conjugates.

U.S. Pat. No. 5,514,363 Method for radiolabeling antibody fragments.

U.S. Pat. No. 5,482,698 Detection and therapy of lesions withbiotin/avidin polymer conjugates.

U.S. Pat. No. 5,443,953 Preparation and use of immunoconjugates.

U.S. Pat. No. 5,439,665 Detection and treatment of infectious andinflammatory lesions.

U.S. Pat. No. 5,364,612 Detection of cardiovascular lesions.

U.S. Pat. No. 5,334,708 Method for radiolabeling monovalent antibodyfragments.

U.S. Pat. No. 5,332,567 Detection and treatment of infections withimmunoconjugates.

U.S. Pat. No. 5,328,679 Methods for technetium/rhenium labeling ofproteins.

U.S. Pat. No. 5,128,119 Methods for technetium/rhenium labeling off(ab′)₂ fragments.

U.S. Pat. No. 5,120,525 Radiolabeled antibody cytotoxic therapy ofcancer.

U.S. Pat. No. 5,101,827 Lymphographic and organ imaging method and kit.

U.S. Pat. No. 5,061,641 Method for radiolabeling proteins.

U.S. Pat. No. 4,932,412 Intraoperative and endoscopic tumor detectionand therapy.

U.S. Pat. No. 4,925,648 Detection and treatment of infectious andinflammatory lesions.

U.S. Pat. No. 4,900,684 CEA immunoassay free of human anti-mouseantibody false positives.

U.S. Pat. No. 4,824,659 Antibody conjugates.

U.S. Pat. No. 4,792,521 Non-enzymatic immunohistochemical stainingsystem and reagents.

U.S. Pat. No. 4,737,453 Sandwich immunoassay utilizing a separationspecific binding substance.

U.S. Pat. No. 4,735,210 Lymphographic and organ imaging method and kit.

U.S. Pat. No. 4,699,880 Method of producing monoclonal anti-idiotypeantibody.

U.S. Pat. No. 4,680,338 Bifunctional linker.

U.S. Pat. No. 4,624,846 Method for enhancing target specificity ofantibody localization and clearance of non-target diagnostic andtherapeutic principles.

U.S. Pat. No. 4,595,654 Method for detecting immune complexes in serum.

XIII.C. Published PCT Patent Applications

WO 02/12347, Immunotherapy for Chronic Myelocytic Leukemia

WO 02/08293, Multivalent Target Binding Protein

WO 02/02150, Stable Radioiodine Conjugates And Methods For TheirSynthesis.

WO 01/97855, Targeted Combination Immunotherapy Of Cancer And InfectiousDiseases.

WO 00/74718, Immunotherapy Of Autoimmune Disorders Using AntibodiesWhich Target B-Cells.

WO 00/67795, Immunotherapy Of B-Cell Malignancies Using Anti-Cd22Antibodies.

WO 00/33874, Boron Neutron Capture Therapy Using Pre-Targeting Methods.

WO 00/21573, Site-Specific Labeling Of Disulfide-Containing TargetingVectors.

WO 00/16808, Methods And Compositions For Increasing The Target-SpecificToxicity Of A Chemotherapy Drug.

WO 00/14537, Diagnosis Of Multidrug Resistance In Cancer And InfectiousLesions.

WO 99/66951, Use Of Bi-Specific Antibodies For Pre-Targeting DiagnosisAnd Therapy.

WO 99/59633, Therapeutics Using A Bispecific Anti-HLA Class Ii InvariantChain X Anti-Pathogen Antibody.

WO 99/56792, Positron Emission Tomography Using Gallium-68 Chelates.

WO 99/46389, Recombinant Onconase, And Chemical Conjugates And FusionProteins Of Recombinant Onconase.

WO 99/30745, Dota-Biotin Derivatives.

WO 99/24472, Glycosylated Antibodies And Antibody Fragments HavingReactive Ketone Groups.

WO 99/11590, Fluorination Of Proteins And Peptides For F-18 PositronEmission Tomography.

WO 99/11294, Stable Radioiodine Conjugates And Methods For TheirSynthesis.

WO 98/50435, Immunotoxins, Comprising An One Protein, Directed AgainstMalignant Cells.

WO 98/42378, Immunotherapy Of B-Cell Malignancies Using Anti-CD22Antibodies.

WO 98/34957, Stimulation Of An Immune Response With Antibodies LabeledWith The ±-Galactosyl Epitope.

WO 98/16254, Non-Antigenic Toxin-Conjugate And Fusion Protein OfInternalizing Receptor System.

WO 98/08548, Stable Radioiodine Conjugates And Methods For TheirSynthesis WO 98/04917, Boron Neutron Capture Therapy Using Pre-TargetingMethods Immunomedics Inc.

WO 98/04293, Improved Detection And Therapy Of Lesions WithBiotin-Chelate Conjugates.

WO 98/02192, Radiometal-Binding Peptide Analogues.

WO 97/41898, Targeted Combination Immunotherapy Of Cancer.

WO 97/40384, Mass Spectrometry And X-Ray Crystallization Analysis OfBiological Material Via Solid Phase Support.

WO 97/34636, Humanization Of An Anti-Carcinoembryonic AntigenAnti-Idiotype Antibody And Use As A Tumor Vaccine And For TargetingApplications.

WO 97/34632, Glycosylated Humanized B-Cell Specific Antibodies.

WO 97/23237, Use Of Immunoconjugates To Enhance The Efficacy OfMulti-Stage Cascade Boosting Vaccines.

WO 97/11370, Recombinant Proteins Having Multiple Disulfide Bonds AndThiol-Substituted Conjugates Thereof.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other documents.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

EXAMPLES

Reagents

Mono-DPTA Peptides

IMP 233 Ac-Phe-Gln-Tyr-Lys(DTPA)-NH₂ Described herein

IMP 240 Ac-Lys(DPTA)-Cys-NH₂ Described herein

Di-DPTA Peptides

IMP 156 Ac-Phe-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂ See Boerman et al.,Pretargeting of renal cell carcinoma: improved tumor targeting with abivalent chelate, Cancer Res. 59:4400-5, 1999.

IMP 192 Ac-Lys(DTPA)-Tyr-Lys(DTPA)-Lys(TSCG-Cys)-NH₂ See Karacay et al.,Experimental pretargeting studies of cancer with a humanized anti-CEA xmurine anti-[In-DTPA] bispecific antibody construct and a(99m)Tc-/(188)Re-labeled peptide, Bioconjug Chem. 11:842-54, 2000.

IMP 222 Ac-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂ Described herein

IMP 240 Ac-Lys(DTPA)-Cys-NH₂ Described herein

Tetra-DPTA Peptides

IMP 246 [Ac-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂]₂ (aka [IMP 222]₂) Describedherein

Bi-Specific Antibodies

hMN-14IgG-(734scFV)₂

-   -   See published PCT Application WO 99/66951 by Hansen et al.,        entitled “Use of Bi-specific Antibodies for Pre-targeting        Diagnosis and Therapy.”

hMN-14IgG^((1253A))-(734scFv)₂

-   -   This mutant derivative of hMN-14IgG-(734scFv)₂, which has a        substitution of its 253rd amino acid residue from isoleucine to        alanine, is described in U.S. Provisional Application Ser. No.        60/361,037 (Atty Docket No. 018733-1037), which was filed Mar.        1, 2002, and is entitled “Bispecific Antibody Point Mutations        for Enhancing Rate of Clearance.”

Example 1 Synthesis of Mono-DTPA peptide IMP 233

IMP 233 Ac-Phe-Gln-Tyr-Lys(DTPA)-NH₂

The IMP 233 peptide was synthesized by solid phase peptide synthesisusing the Fmoc strategy and Rink Amide Resin (0.25 g, 0.8 mmol/gsubstitution). The protected amino acids Fmoc-Lys(Aloc)-OH,Fmoc-Tyr(But)-OH, Fmoc-Gln(Trt)OH, Fmoc-Phe-OH and Acetic anhydride wereadded in that order to the resin.

The Aloc side chain protecting group was removed and the DTPAtetra-t-butyl ester was added. The peptide was cleaved and purified byHPLC to afford 0.009 g of the purified peptide (ESMS MH⁺1002).

Example 2 Synthesis of Di-DTPA Peptide IMP 156

IMP 156 Ac-Phe-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂

The IMP 156 peptide was synthesized by solid phase peptide synthesisusing the Fmoc method and Rink Amide Resin (or Sieber amide resin). Theprotected amino acids; Fmoc-Lys(Aloc)-OH, Fmoc-Tyr(But)-OH,Fmoc-Lys(Aloc)OH, Fmoc-Phe-OH and Acetic anhydride were added in thatorder to the resin.

The Aloc side chain protecting groups were removed and the DTPAtetra-t-butyl ester was added. The peptide was cleaved and purified byHPLC (ESMS MH⁺1377).

Example 3 Synthesis of Di-DTPA Peptide IMP 222

IMP 222 Ac-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂

The IMP 222 peptide was synthesized by solid phase peptide synthesisusing the Fmoc and Rink Amide Resin (0.25 g, 0.8 mmol/g substitution).The protected amino acids; Fmoc-Lys(Aloc)-OH, Fmoc-Tyr(But)-OH,Fmoc-Lys(Aloc)-OH, Fmoc-Cys(Trt)-OH and Acetic anhydride were added inthat order to the resin. The Aloc side chain protecting groups wereremoved and the DTPA tetra-t-butyl ester was added. The peptide wascleaved and purified by HPLC to afford 0.125 g of the purified peptide(ESMS MH+1333).

Example 4 Synthesis of Tetra-DTPA Peptide IMP 246

IMP 246 [Ac-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂]₂

The peptide, 0.0531 G (IMP 222, 3.98×10⁻⁵ MOL,AC-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂) was dissolved in a solution whichcontained 1.0 mL DMSO, 0.2 mL Diisopropylethylaminie, and 0.3 mL water.The solution was incubated at room temperature for four days and thenpurified by reverse phase HPLC to afford 0.0336 g of the disulfide (ESMSMH⁺2663).

Example 5 IMP 246 Kits

The peptide (0.0022 g) was dissolved in 100 mL of a solution thatcontained 0.418 g citric acid and 10.06 g of HPCD buffered at pH 4.3.The solution was sterile filtered through a 0.22 μM Millex GV filter in1 mL aliquots into vials which were immediately frozen and lyophilized.

Example 6 In-111 Labeling of IMP 246

The In-111 (0.4 mCi) was diluted with 0.5 mL water and added to alyophilized IMP 246 kit. The solution was incubated at room temperaturefor 10 min. A 1.5 mL aliquot of a solution containing 2.56×10⁻⁵ M Indiumin 0.5 M NaOAc Buffer pH 7.17 was then added to the kit.

Example 7 Evaluation of Affinity of di-DTPA Peptide IMP 192 ComplexesComprising hMN-141gG^((1253A))-(734scFv)₂ by HPLC

The binding of In-DTPA peptides to the anti-In-DTPA antibody hMN-141g^((1253A))-(734scFV)₂ was examined by size exclusion HPLC. The bsMAbwas radiodinated using chloramine T (Greenwood and Hunter). Binding ofthe radioiodinated bsMAbs to CEA, W12 (rat anti-MN-14 idiotypicantibody) and radiolabeled peptidyl DTPA chelate was examined onanalytical size exclusion HPLC. Approximately 90% of the radioidinatedbsMAb bound to CEA upon treatment with 10-20×molar excess of CEA. ThebsMAb complexed with radiolabeled indium-DTPA chelates (IMP-156 orIMP-192).

An IMP 192 kit was labeled with Tc-99m 20.9 mCi. Aliquots from the kitwere diluted and mixed with hMN-14IgG^((1253A))-(734scFV)₂ in thefollowing molar ratios (peptide/Ab) 1:5, 1:1, and 20:1. Thepeptide/antibody mixtures, the peptide alone and the antibody alone wereexamined on a Bio-Sil SEC 250 300 mm×7.8 mm HPC column eluted at 1mL/min with 0.2 M phosphate buffer pH 6.8.

The HPLC traces (FIGS. 3-7) show that essentially only onepeptide/antibody complex is formed. A known standard ofhMN-14IgG^((1253A))-(734scFV)₂ eluted from the column at about 9.41minutes (FIG. 3). A known standard of Tc-99m IMP 192 eluted from thecolumn at about 14.85 minutes (FIG. 4). When a 1:1 mixture ofhMN-14IgG^((1253A))-(734scFV)₂ to Tc-99m IMP 192 was applied to thecolumn, only one peak was observed at about 9.56 minutes (FIG. 5). Incontrast, when a 1:5 mixture of hMN-14IgG^((1253A))-(734scFV)₂ to Tc-99mIMP 192 was applied to the column, two major peaks were observed, one atabout 9.56 minutes [hMN-14IgG^((1253A))-(734scFV)₂] and the other atabout 14.80 minutes (Tc-99m IMP 192) (FIG. 6). When a 20:1 mixture ofhMN-14 IgG^((1253A))-(734scFv)₂ to Tc-99m IMP 192 was applied to thecolumn, only one peak was observed at 9.56 minutes (FIG. 7).

Example 8 Stoichiometry of Targetable Complexes

This Example describes experiments designed to determine thestoichiometry of different species of targetable complexes that arisewhen the targetable divalent construct IMP 246 is mixed with bsAbshMN-14IgG^((1253A))-(734scFV)₂, and hMN-14IgG-(734scFV)₂.

The peptide IMP 246 was dissolved in 100 mL of a solution whichcontained 0.418 g citric acid and 10.06 g of HPCD buffered at pH 4.3.The solution was filter sterilized through a 0.22 μm Millex GV filtersin 1 mL aliquots into vials that were immediately frozen andlyophilized.

The IMP 246 was labeled with In-111 as follows. The In-111 was dilutedwith 0.5 mL water and added to a lyophilized IMP 246 kit. The solutionwas allowed to incubate at room temperature for 10 min. A 1.5 mL aliquotof a solution containing 2.56×10-5 Indium in 0.5 M NaOAc buffer pH 7.17was then added to the kit.

The In-111-labelled IMP 246 was mixed with bsAb in a 1:10 (IMP 246:bsAb)mole ratio and then examined by size exclusion HPLC. The results areshown in FIG. 8. When mixed with the mutant bsAb, i.e.,hMN-14IgG^((1253A))-(734scFV)₂, about 90% of the In-111 label was foundat a clean sharp peak at 8.5 min (FIG. 8A). When mixed withhMN-14IgG-(734scFv)₂, the peak was somewhat broader, comprised about 83%of the In-111 label and was found at 7.5 min (FIG. 8B). In contrast, theantibody:peptide complexes formed when IMP 192 was used were found at9.5 min (FIG. 7).

Example 9 Synthesis of Mono-DTPA Peptide IMP 240 IMP 240Ac-Lys(DTPA)-Cys-NH₂

The Tetra t-butyl ester of DTPA (J Med Chem 1996 Aug. 30;39(18):3451-60,Reassessment of diethylenetriaminepentaacetic acid (DTPA) as a chelatingagent for indium-111 labeling of polypeptides using a newly synthesizedmonoreactive DTPA derivative. Arano Y, Uezono T, Akizawa H, Ono M,Wakisaka K, Nakayama M, Sakahara H, Konishi J, Yokoyama A.) 1.424 g wasdissolved in 5.5 mL dioxane. N-Hydroxysuccinimide, 0.304 g was addedfollowed by 0.4 mL of diisopropylcarbodiimide (DIC) and mixed for 1 hrat room temperature. The remaining reagents 1.049 g Na₂CO₃ and 0.862 gAc-Lys-OH were mixed then added 5 mL water was added. Thelysine/carbonate solution was then mixed with the activated DTPAreagent. The reaction was stirred at room temperature overnight and thenquenched with 20 mL 1 M citric acid. The citric acid solution wasextracted with 2×50 mL portions of chloroform, dried over Na₂SO₄ andconcentrated under reduced pressure to obtain 2.031 g of crude product.The crude product was dissolved in 6 mL dioxane and mixed with 0.264 gN-Hydroxysuccinimide and 0.38 mL DIC. The reaction was mixed at roomtemperature for 2.5 hr then 0.414 g H-Cys(Trt)-NH₂ was added along with0.3 mL diisopropylethylamine. The reaction was stirred at roomtemperature overnight and then filtered. The solids were washed withdioxane and the filtrates were combined. The crude product wasconcentrated under reduced pressure. The crude product was treated witha cleavage solution of 25 mL Trifluoro acetic acid, 1 mLtriisopropylsilane and 1 mL anisole. The cleavage reaction mixture waspoured into 2×40 mL ether after 3.5 hr. The peptide was collected bycentrifugation. The precipitated peptide was washed with 3×30 mL ether.The precipitate was dried then purified by reverse phase HPLC to obtain0.2152 g of the desired product MNa+688.

Example 10 Binding Studies Using Surface Plasmon Resonance

The binding of In-DTPA peptides to the anti-In-DTPA antibodyhMN-14IgG^((1253A))-(734scFv)₂ was examined by affinity blocking studiesusing the IMP 240 peptide, Ac-Lys(DPTA)-Cys-NH₂, which comprises asingle DPTA group and a single Cys residue that can be used to formdisulfide bridges with a second molecule having a free sulfhydryl group.

Preparation of IMP 240-coated BIACore Chip

The binding studies were performed on a chip coated with IMP 240 usingthe disulfide connection as recommended by BIAcore. The chip surface wasregenerated after each assay with 100 μL 0.025 M In-DTPA inHBS-In-citrate buffer as described. The binding studies were done withpicomoles of antibody therefore binding was very sensitive to theslightest trace of In-DTPA left after the displacement wash.

IMP 156 Binding to hMN-14IgG^((1253A))-(734scFV)₂

The affinity studies show that when IMP 156 was mixed withhMN-14IgG^((1253A))-(734scFv)₂ in a 1:1 ratio the antibody binding tothe IMP 240 chip was blocked. This indicates that both 734 binding siteswere filled with the two DTPA's on a single peptide.

IMP 233 Binding to hMN-14 IgG_((1253A))-(734scFV)₂

The affinity studies show the binding of the antibody was not completelyblocked even when four equivalents of the mono-DTPA peptide werepremixed with the antibody. Although not wishing to be bound by anyparticular theory, this is probably due to the dissociation of a portionof the mono-DTPA peptide from the antibody during the test.

Binding Studies of IMP 156 Binding to c734 IgG

Studies were performed with c734-IgG to compare the In-DTPA peptidebinding behavior of this IgG to the hMN-14IgG^((1253A))-(734scFV)₂. Itwas necessary to add two or more equivalents of IMP 156 to block thebinding of c734 to the chip whereas the hMN-14IgG^((1253A))-(734scFV)₂was blocked by one equivalent of IMP 156.

This indicates that both In/DTPA's of IMP 156 were simultaneously boundto both scFv's of hMN-14IgG^((1253A))-(734scFV)₂ whereas only oneIn/DTPA of IMP 156 was bound to the binding arm of c734-IgG and anotherIn/DTPA on another molecule of IMP 156 was needed to block the otherbinding arm of c734-IgG.

Binding Studies of IMP 233 Binding to c734 IgG

The affinity studies showed that even 4:1 IMP 233/c734 IgG did notcompletely block the binding of the antibody to the IMP 240 chip.

This experiment demonstrated that it was difficult to completely blockthe binding of the binding of c734 IgG to the IMP 240 chip with apeptide bearing a singe In-DTPA (IMP 233). This meant that either therewas a certain amount of free c734 IgG (or at least one binding armavailable for binding) or the In-DTPA on the chip was able to displacethe In-DTPA of IMP 233. The fact that IMP 156 completely blocked thebinding of hMN-14IgG^((1253A))-(734scFV)₂ (when mixed in a 1:1 ratioPeptide: Antibody) to the IMP 240 chip demonstrated that both of theDTPA binding sites were filled and that the affinity of thehMN-14IgG^((1253A))-(734scFV)₂ for in2-IMP 156 was far higher than theaffinity of a single In-DTPA for a c734 IgG binding arm.

Example 11 Attachment of IMP 222 to a BIAcore Chip

The CM-5 Chip has a carbohydrate attached to a gold surface which hasbeen derivitized with carboxylic acids. The carboxylic acids on thesurface were activated with NHS(N-hydroxysuccinimide) and a watersoluble carbodiimide, EDC (N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride) essentially according to themanufacturer's instructions (see Application Note 9, BiacoreInternational AB, Uppsala, Sweden, published on-line athttp://www.biacore.com/products/pdf/ANLigandImmobilization. pdf).

A solution of 2-(2-pyridinylthio)ethaneamine hydrochloride (PDEA) in 0.1M pH 8.5 borate buffer was then added essentially according to themanufacturer's instructions. The IMP 222 peptide,Ac-Cys-Lys(DTPA)-Tyr-Lys(DTPA)-NH₂, was dissolved in 0.01 M pH 4.3formate buffer then added to the chip as described.

The peptide forms a disulfide linkage to the chip when added in thismanner. Finally, the remaining unreacted active ester and PDEA arequenched by the addition of a solution containing cysteine and NaCl in0.1 M formate buffer pH 4.3.

Example 12 Serum Stability

Stability of bsAb in Serum

Bi-specific antibody (bsAb) was radioiodinated and tested for stabilityin fresh human serum at 37° C. under a humidified 5% CO₂ atmosphere.Aliquots were examined on SE-HPLC. In order to detect radioiodineassociated with serum proteins, the aliquots were mixed with WI2 toshift the bsMAb peak to earlier retention times. The bsMAbs showed about3-5% loss of binding capacity to WI2 after 48 h incubation in serum.Slight aggregate formation (about 4-7%) was observed upon incubation ofthe bsMAbs in serum for 72 hours.

Stability of Targetable Constructs in Serum

A 40 μL aliquot of the labeled peptide was diluted with 400 μL of freshmouse serum and incubated at 37° C. for 17.5 hours. A 10 μL aliquot wasremoved and mixed with 5 μL of 3.2 mg/mL 14IgG^((1253A))-(734scFv)₂ anddiluted with 40 μL water. A 10 μL aliquot of the diluted mixture wasthen examined by size exclusion HPLC.

Stability of Targetable Complexes in Serum

Peptide Plus hMN-14 IgG^((1251A))-(734scFV)₂ Serum Stability:

A 4 μL aliquot of the labeled peptide solution was mixed with 10 μL ofthe 3.2 mg/mL hMN-14IgG^((1253A))-(734scFv)₂ and diluted in 400 μL offresh mouse serum. The serum sample was incubated at 37° C. for 16 hrand then analyzed by size exclusion HPLC.

The IMP 246 peptide was unstable in mouse serum after 16 hours, asdemonstrated by the presence of several overlapping peaks in the HPLCtracing. Three broad peaks comprised about 20%, 41% and 37% of the areaunder the curve of the HPLC tracing. In contrast, the stability of IMP246 was greatly increased in the presence of the bi-specific antibody,as ≧90% of the area under the curve of the HPLC tracing was found in adistinct peak at the predicted position.

Example 13 Evaluation of Complexes Comprising hA20-IgG-(734scFv)₂ invitro

hA20-IgG-(734scFv)₂ is made using methods described in PCT ApplicationPublication No. WO 99/66951 by Hansen et al., with the only change beingthat cDNA coding for the variable chains of hA20 is used in place ofcDNA coding for hMN-14 variable chains. The cDNA coding for the constantregions of both hMN-14 and hA20 are identical, as is the cDNA coding forthe linker and scFv of monoclonal antibody 734.

Raji cells in culture are incubated with hA20IgG-(734scFv)₂. To one setof wells is added IMP-246 to cross-link molecules of hA20IgG-(734scFv)₂bound to CD20 on the surface of the Raji cells. A second set of wells towhich IMP-246 is not added serve as controls, and both sets of wellsincubated at 37 degrees C. After 3 days, the cells to which IMP-246 wasadded are determined to have undergone extensive apoptosis. Minimalapoptosis was observed in the control wells to which IMP-246 was notadded.

An average of 5 molecules IMP-222 are conjugated to human serum albumin(hAlb-222). The experiment described in the paragraph above is repeated,but hAb-222 is used to cross-link molecules of hA20IgG-(734scFv)₂ boundto CD20 on the surface of the Raji cells, in place of IMP-246. Extensiveapoptosis of cells is observed in wells to which hAlb-222 was added.

Illustrative Embodiments

Additional embodiments are within the scope of the invention. Forexample, the invention is further illustrated by the following numberedembodiments:

1. A targetable construct comprising (i) a molecular scaffold and (ii)two pairs of a carrier epitope, wherein said targetable construct, whencombined with a bi-specific antibody comprising (i) two copies of afirst arm comprising a binding site for said carrier epitope, and (ii)two copies of a second arm comprising a binding site for a targetepitope, forms a targetable complex, wherein one or more of thefollowing applies:

-   -   (a) said targetable complexes have a Kd for said target epitope        from about 0.1 nM to about 100 nM,    -   (b) mixing said targetable construct and said bi-specific        antibody at relative concentrations ranging from about 10⁻³ to        about 10³ results in a mixture in which greater than about 75%        of the complexes therein have a defined stoichiometry of two        molecules of said bi-specific antibody, and one molecule of said        targetable construct, and    -   (c) a pair of carrier epitopes is simultaneously bound by said        two copies of a first arm comprising a binding site for said        carrier epitope, wherein said two copies of a first arm        comprising a binding site for said carrier epitope are part of        said bi-specific antibody.

2. The targetable construct of embodiment 1, wherein mixing saidtargetable construct and said bi-specific antibody at relativeconcentrations ranging from about 10⁻³ to about 10³ results in a mixturein which greater than about 85% of the multimeric complexes have adefined stoichiometry of two molecules of said bi-specific antibody, andone molecule of said targetable construct.

3. The targetable construct of embodiment 1, wherein mixing saidtargetable construct and said bi-specific antibody at relativeconcentrations ranging from about 10⁻³ to about 103 results in a mixturein which greater than about 95% of the multimeric complexes have adefined stoichiometry of two molecules of said bi-specific antibody, andone molecule of said targetable construct.

4. The targetable construct of embodiment 1, wherein mixing saidtargetable construct and said bi-specific antibody at relativeconcentrations ranging from about 10⁻³ to about 10³ results in a mixturein which greater than about 99% of the multimeric complexes have adefined stoichiometry of two molecules of said bi-specific antibody andone molecule of said targetable construct.

5. The targetable construct of embodiment 1, wherein said Kd for saidtarget epitope is from about 0.1 nM to 10 nM.

6. The targetable construct of embodiment 5, wherein said Kd for saidtarget epitope is from about 0.5 nM to about 5 nM.

7. The targetable construct of embodiment 6, wherein said Kd for saidtarget epitope is about 1 nM.

8. The targetable construct of embodiment 1, wherein said molecularscaffold is a peptide or peptide derivative.

9. The targetable construct of embodiment 1, wherein said targetableconstruct is IMP 246.

10. The targetable construct of embodiment 1, wherein said targetableconstruct comprises two constructs that are conjugated to each other,wherein each construct comprises a molecular scaffold and a pair ofcarrier epitopes.

11. The targetable construct of embodiment 10, wherein each of saidconstructs is independently selected from the group consisting of IMP156, IMP 192 and IMP 222.

12. The targetable construct of embodiment 1, wherein said carrierepitope is a hapten.

13. The targetable construct of embodiment 1, wherein said carrierepitope is a chelator, or a complex between a chelator and a metal ion.

14. The targetable construct of embodiment 13, wherein said chelator isselected from the group consisting of DTPA, DOTA, benzyl DTPA, NOTA, andTETA.

15. The targetable construct of embodiment 1, wherein said bi-specificantibody is [IgG]-[scFv]2; wherein IgG is a human, chimeric orCDR-grafted antibody; further wherein scFv is a human, chimeric orCDR-grafted single chain antibody specific for a hapten; and furtherwherein said scFv is extended from the carboxyl terminal amino acid ofthe heavy chains of said IgG by a linker peptide.

16. The targetable construct of embodiment 15, wherein said bi-specificantibody is selected from the group consisting of[hMN14-IgG1]-[734scFv]₂ and [hMN14-IgG1^((1253A))]-[734scFV]₂.

17. The targetable construct of embodiment 15, wherein said bi-specificantibody is selected from the group consisting of[hMN14-IgG1]-[679scFv]₂ and [hMN14-IgG1^((1253A))]-[679scFv]₂.

18. The targetable construct of embodiment 15, wherein said bi-specificantibody is selected from the group consisting of [hA20-IgG1]-[734scFv]₂and [hA20-IgG1^((1253A))]-[734scFv]₂.

19. The targetable construct of embodiment 15, wherein said bi-specificantibody is selected from the group consisting of [hA20-IgG1]-[679scFv]₂and [hA20-IgG1^((1253A))]-[679scFv]₂.

20. The targetable construct of embodiment 15, wherein said bi-specificantibody is selected from the group consisting of [hLL2-IgG1]-[734scFv]₂and [hLL2-IgG1^((1253A))]-[734scFv]₂.

21. The targetable construct of embodiment 15, wherein said bi-specificantibody is selected from the group consisting of [hLL2-IgG1]-[679scFv]₂and [hLL2-IgG1^((1253A))]-[670scFv]₂.

22. The targetable construct of embodiment 1, wherein said targetableconstruct further comprises a bioactive moiety.

23. The targetable construct of embodiment 22, wherein said bioactivemoiety is selected from the group consisting of a drug, a prodrug, anenzyme, a hormone, an immunomodulator, an oligonucleotide; aradionuclide, an image enhancing agent and a toxin.

24. The targetable construct of embodiment 23, wherein said enzyme isselected from the group consisting of malate dehydrogenase,staphylococcal nuclease, delta-V-steroid isomerase, yeast alcoholdehydrogenase, α-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, α-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

25. The targetable construct of embodiment 23, wherein saidimmunomodulator is selected from the group consisting of a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic growth factor, acolony stimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof.

26. The targetable construct of embodiment 23, wherein saidimmunomodulator consists essentially of IL-1, IL-2, IL-3, IL-6, IL-10,IL-12, IL-18, IL-21, G-CSF, GM-CSF, interferon-γ, -α, -β or -γ, TNF-α,and “S1 factor.

27. The targetable construct of embodiment 23, wherein saidoligonucleotide is an anti-sense oligonucleotide.

28. The targetable construct of embodiment 27, wherein said anti-senseoligonucleotide is bcl-2 or p53.

29. A solid support to which the targetable construct of embodiment 1 isbound.

30. A biosensor comprising the targetable construct of embodiment 1.

31. A targetable construct comprising (i) a molecular scaffold and (ii)two pairs of carrier epitopes, wherein the first of said two pairs ofcarrier epitopes is specifically bound by a first bi-specific antibody,and the second of said two pairs of carrier epitopes is specificallybound by a second bi-specific antibody, wherein said targetableconstruct forms a targetable complex when combined with

-   -   (i) a first bi-specific antibody, said first bi-specific        antibody copmrising (a) two copies of a first arm comprising a        binding site for said carrier epitope, and (b) two copies of a        second arm comprising a binding site for a first target epitope,        and    -   (ii) a second bi-specific antibody, said second bi-specific        antibody comprising (a) two copies of a first arm comprising a        binding site for said carrier epitope, and (b) two copies of a        second arm comprising a binding site for a second target        epitope;    -   wherein said first bi-specific antibody and said second        bi-specific antibody can be the same or different, said pairs of        carrier epitopes can be the same or different, and said target        epitopes can be the same or different, and wherein one or more        of the following applies:    -   (a) said targetable complex has a Kd of from about 0.1 nM to        about 100 nM for either or both of said target epitopes,    -   (b) mixing said targetable construct and said bi-specific        antibodies at relative concentrations ranging from about 10⁻³ to        about 10³ results in a mixture in which greater than about 75%        of the multimeric complexes have a defined stoichiometry of one        molecule of said first bi-specific antibody, one molecule of        said second bi-specific antibody, and one molecule of said        targetable construct; and    -   (c) a pair of carrier epitopes is simultaneously bound by said        two copies of a first arm comprising a binding site for said        carrier epitope, wherein said two copies of a first arm        comprising a binding site for said carrier epitope are part of        said bi-specific antibody.

32. The targetable construct of embodiment 31, wherein said pairs ofcarrier epitopes are different, but said target epitopes are the same.

33. The targetable construct of embodiment 31, wherein said firstantibody and said second antibody are different, but said targetepitopes are the same.

34. The targetable constrict of embodiment 31, wherein said carrierepitopes are the same, and said first arm comprising a binding site forsaid carrier epitope of said first bi-specific antibody and said firstarm comprising a binding site for said carrier epitope of said secondbi-specific antibody are the same, but said first target epitope andsaid second target epitope are not the same.

35. The targetable construct of embodiment 31, wherein mixing saidtargetable construct and said bi-specific antibodies at relativeconcentrations ranging from about 10⁻³ to about 10³ results in a mixturein which greater than about 85% of the multimeric complexes have adefined stoichiometry of two molecules of said bi-specific antibody orantibody derivative and one molecule of said targetable construct.

36. The targetable construct of embodiment 31, wherein mixing saidtargetable construct and said bi-specific antibody at relativeconcentrations ranging from about 10⁻³ to about 10³ results in a mixturein which greater than about 95% of the multimeric complexes have adefined stoichiometry of two molecules of said bi-specific antibody orantibody derivative and one molecule of said targetable construct.

37. The targetable construct of embodiment 31, wherein mixing saidtargetable construct and said bi-specific antibody at relativeconcentrations ranging from about 10⁻³ to about 10³ results in a mixturein which greater than about 99% of the multimeric complexes have adefined stoichiometry of two molecules of said bi-specific antibody orantibody derivative and one molecule of said targetable construct.

38. The targetable construct of embodiment 31, wherein said Kd for saidtarget epitope is from about 0.1 nM to 10 nM.

39. The targetable construct of embodiment 38, wherein said Kd for saidtarget epitope is from about 0.5 nM to about 5 nM.

40. The targetable construct of embodiment 39, wherein said Kd for saidtarget epitope is about 1 nM.

41. The targetable construct of embodiment 31, wherein said targetableconstruct comprises two constructs that are conjugated to each other,wherein each construct comprises a molecular scaffold and a pair ofcarrier epitopes.

42. The targetable construct of embodiment 41, wherein each of saidconstructs is independently selected from the group consisting of IMP156, IMP 192 and IMP 222.

43. The targetable construct of embodiment 31, wherein said molecularscaffold is a peptide or peptide derivative.

44. The targetable construct of embodiment 31, wherein said carrierepitope is a hapten.

45. The targetable construct of embodiment 31, wherein said carrierepitope is a chelator, or a complex between a chelator and a metal ion.

46. The targetable construct of embodiment 45, wherein said chelator isselected from the group consisting of DTPA, DOTA, benzyl DTPA, NOTA, andTETA.

47. The targetable construct of embodiment 31, wherein said targetableconstruct further comprises a bioactive moiety.

48. The targetable complex of embodiment 47, wherein said bioactivemoiety is selected from the group consisting of a drug, a prodrug, anenzyme, a hormone, an immunomodulator, an oligonucleotide, aradionuclide, an image enhancing agent and a toxin.

49. The targetable construct of embodiment 48, wherein said enzyme isselected from the group consisting of malate dehydrogenase,staphylococcal nuclease, delta-V-steroid isomerase, yeast alcoholdehydrogenase, α-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, β-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

50. The targetable construct of embodiment 48, wherein saidimmunomodulator is selected from the group consisting of a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic growth factor, acolony stimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof.

51. The targetable construct of embodiment 48, wherein saidimmunomodulator is selected from the group consisting of IL-1, IL-2,IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, G-CSF, GM-CSF, interferon-γ, -α,-β or -γ, TNF-α, and “S1 factor.

52. The targetable construct of embodiment 48, wherein saidoligonucleotide is an anti-sense oligonucleotide.

53. The targetable construct of embodiment 52, wherein said anti-senseoligonucleotide is bcl-2 or p53.

54. The targetable construct of embodiment 34, wherein said first armcomprising a binding site for said carrier epitope of said firstbi-specific antibody, and said first arm comprising a binding site forsaid carrier epitope of said second bi-specific antibody, are both[734scFv]₂.

55. A solid support to which the targetable construct of embodiment 31is bound.

56. A biosensor comprising the targetable construct of embodiment 31.

57. A targetable construct comprising a molecular scaffold and X pairsof carrier epitopes, wherein each of said pairs of carrier epitopes isspecifically bound by one of Xbi-specific antibodies, each bi-specificantibody comprising (a) two copies of a first arm comprising a bindingsite for a carrier epitope, and (b) two copies of a second armcomprising a binding site for one of Y target epitopes, and wherein

-   -   (i) X is a whole integer ≧3,    -   (ii) Y is a whole integer ≧1,    -   (iii) said Xbi-specific antibodies can be the same or a mixture        of different bi-specific antibodies,    -   (iv) said Xpairs of carrier epitopes can be the same or a        mixture of different carrier epitopes, and    -   (v) when Y≧2, said Y target epitopes can be the same or a        mixture of different target epitopes,    -   and wherein one or more of the following applies:    -   (a) said targetable complex has a Kd of from about 0.1 nM to        about 100 nM for at least one of said target epitopes,    -   (b) a pair of carrier epitopes is simultaneously bound by said        two copies of a first arm comprising a binding site for said        carrier epitope, wherein said two copies of a first arm        comprising a binding site for said carrier epitope are part of        said bi-specific antibody.

58. The targetable construct of embodiment 57, wherein said Kd for atleast one of said target epitopes is from about 0.1 nM to 10 nM.

59. The targetable construct of embodiment 58, wherein said Kd for atleast one of said target epitopes is from about 0.5 nM to about 5 nM.

60. The targetable construct of embodiment 59, wherein said Kd for forat least one of target epitope is about 1 nM.

61. The targetable construct of embodiment 57, wherein said molecularscaffold is a peptide or peptide derivative.

62. The targetable construct of embodiment 57, wherein at least one ofsaid carrier epitopes is a hapten.

63. The targetable construct of embodiment 57, wherein said targetableconstruct further comprises a bioactive moiety.

64. The targetable construct of embodiment 63, wherein said bioactivemoiety is selected from the group consisting of a drug, a prodrug, anenzyme, a radionuclide, an image enhancing agent and a toxin.

65. The targetable construct of embodiment 64, wherein said enzyme isselected from the group consisting of malate dehydrogenase,staphylococcal nuclease, delta-V-steroid isomerase, yeast alcoholdehydrogenase, α-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, β-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

66. The targetable construct of embodiment 64, wherein saidimmunomodulator is selected from the group consisting of a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic growth factor, acolony stimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof.

67. The targetable construct of embodiment 64, wherein saidimmunomodulator is selected from the group consisting of IL-1, IL-2,IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, G-CSF, GM-CSF, interferon-γ, -α,-β or -γ, TNF-α, and “S1 factor.

68. The targetable construct of embodiment 64, wherein saidoligonucleotide is an anti-sense oligonucleotide.

69. The targetable construct of embodiment 68, wherein said anti-senseoligonucleotide is bcl-2 or p53.

70. A solid support to which the targetable construct of embodiment 57is bound.

71. A biosensor comprising the targetable construct of embodiment 57.

72. A targetable complex comprising four arms capable of binding atarget epitope, said tetravalent targetable complex comprising:

-   -   (a) a targetable construct comprising (i) a molecular scaffold        and (ii) two pairs of a carrier epitope; and    -   (b) two molecules of a bi-specific antibody, each antibody        comprising (i) two arms, each arm comprising a binding site for        said carrier epitope, and (ii) two arms, each comprising a        binding site for said target epitope,    -   wherein one or more of the following applies:    -   (I) said targetable complexes have a Kd for said target epitope        from about 0.1 nM to about 100 nM,    -   (II) mixing said targetable construct and said bi-specific        antibody at relative concentrations ranging from about 10⁻³ to        about 10³ results in a mixture in which greater than about 75%        of the complexes therein have a defined stoichiometry of two        molecules of said bi-specific antibody, and one molecule of said        targetable construct, and    -   (III) a pair of carrier epitopes is bound by said bi-specific        antibody in a 1:1 ratio.

73. The targetable complex of embodiment 72, wherein mixing saidtargetable construct and said bi-specific antibody at relativeconcentrations ranging from about 10⁻³ to about 10³ results in a mixturein which greater than about 85% of the multimeric complexes have adefined stoichiometry of two molecules of said bi-specific antibody, andone molecule of said targetable construct.

74. The targetable complex of embodiment 72, wherein mixing saidtargetable construct and said bi-specific antibody at relativeconcentrations ranging from about 10⁻³ to about 10³ results in a mixturein which greater than about 95% of the multimeric complexes have adefined stoichiometry of two molecules of said bi-specific antibody, andone molecule of said targetable construct.

75. The targetable complex of embodiment 72, wherein mixing saidtargetable construct and said bi-specific antibody at relativeconcentrations ranging from about 10⁻³ to about 10³ results in a mixturein which greater than about 99% of the multimeric complexes have adefined stoichiometry of two molecules of said bi-specific antibody, andone molecule of said targetable construct.

76. The targetable complex of embodiment 72, wherein said Kd for saidtarget epitope is from about 0.1 nM to 10 nM.

77. The targetable complex of embodiment 76, wherein said Kd for saidtarget epitope is from about 0.5 nM to about 5 nM.

78. The targetable complex of embodiment 77, wherein said Kd for saidtarget epitope is about 1 nM.

79. The targetable complex of embodiment 72, wherein said molecularscaffold is a peptide or peptide derivative.

80. The targetable complex of embodiment 72, wherein said targetableconstruct is IMP 246.

81. The targetable complex of embodiment 72, wherein said targetableconstruct comprises two constructs that are conjugated to each other,wherein each construct comprises a molecular scaffold and a pair ofcarrier epitopes.

82. The targetable complex of embodiment 81, wherein each of saidconstructs is independently selected from the group consisting of IMP156, IMP 192 and IMP 222.

83. The targetable complex of embodiment 72, wherein said carrierepitope is a hapten.

84. The targetable complex of embodiment 72, wherein said carrierepitope is a chelator, or a complex between a chelator and a metal ion.

85. The targetable complex of embodiment 84, wherein said chelator isselected from the group consisting of DTPA, DOTA, benzyl DTPA, NOTA, andTETA.

86. The targetable complex of embodiment 72, wherein said bi-specificantibody is selected from the group consisting of [hMN]₂-[734scFv]₂ and[hMN^((1253A))]₂-[734scFv]₂.

87. The targetable complex of embodiment 72, wherein at least one armcomprising a binding site for said carrier epitope is 734scFv.

88. The targetable complex of embodiment 72, wherein said targetableconstruct further comprises a bioactive moiety.

89. The targetable complex of embodiment 88, wherein said bioactivemoiety is selected from the group consisting of a drug, a prodrug, anenzyme, a radionuclide, a hormone, an immunomodulator, anoligonucleotide, an image enhancing agent and a toxin. a hormone, animmunomodulator, an oligonucleotide; a radionuclide, an image enhancingagent and a toxin.

90. The targetable construct of embodiment 89, wherein said enzyme isselected from the group consisting of malate dehydrogenase,staphylococcal nuclease, delta-V-steroid isomerase, yeast alcoholdehydrogenase, α-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, β-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

91. The targetable construct of embodiment 89, wherein saidimmunomodulator is selected from the group consisting of a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic growth factor, acolony stimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof.

92. The targetable construct of embodiment 89, wherein saidimmunomodulator is selected from the group consisting of IL-1, IL-2,IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, G-CSF, GM-CSF, interferon-γ, -α,-β or -γ, TNF-α, and “S1 factor.

93. The targetable construct of embodiment 89, wherein saidoligonucleotide is an anti-sense oligonucleotide.

94. The targetable construct of embodiment 93, wherein said anti-senseoligonucleotide is bcl-2 or p53.

95. A solid support to which the targetable complex of embodiment 72 isbound.

96. A biosensor comprising the targetable complex of embodiment 72.

97. A targetable complex, said targetable complex comprising:

-   -   (a) a targetable construct comprising (i) a molecular scaffold        and (ii) two pairs of carrier epitopes, wherein the first of        said two pairs of carrier epitopes is specifically bound by a        first bi-specific antibody, and the second of said two pairs of        carrier epitopes is specifically bound by a second bi-specific        antibody, wherein said targetable construct forms a targetable        complex when combined with    -   (b) a first bi-specific antibody, said first bi-specific        antibody comprising (i) two copies of a first arm comprising a        binding site for said carrier epitope, and (ii) two copies of a        second arm comprising a binding site for a first target epitope,        and    -   (c) a second bi-specific antibody, said second bi-specific        antibody comprising (i) two copies of a first arm comprising a        binding site for said carrier epitope, and (ii) two copies of a        second arm comprising a binding site for a second target        epitope;    -   wherein said first bi-specific antibody and said second        bi-specific antibody can be the same or different, said pairs of        carrier epitopes can be the same or different, and said target        epitopes can be the same or different, and wherein one or more        of the following applies    -   (I) said targetable complexes have a Kd for said target epitope        from about 0.1 nM to about 100 nM,    -   (II) mixing said targetable construct and said bi-specific        antibody at relative concentrations ranging from about 10⁻³ to        about 10³ results in a mixture in which greater than about 75%        of the complexes therein have a defined stoichiometry of two        molecules of said bi-specific antibody, and one molecule of said        targetable construct, and    -   (III) each of said pairs of carrier epitopes is bound by one of        said bi-specific antibodies in a 1:1 ratio.

98. The targetable complex of embodiment 97, wherein mixing saidtargetable construct and said bi-specific antibody at relativeconcentrations ranging from about 10⁻³ to about 10³ results in a mixturein which greater than about 85% of the multimeric complexes have adefined stoichiometry of two molecules of said bi-specific antibody, andone molecule of said targetable construct.

99. The targetable complex of embodiment 97, wherein mixing saidtargetable construct and said bi-specific antibody at relativeconcentrations ranging from about 10⁻³ to about 10³ results in a mixturein which greater than about 95% of the multimeric complexes have adefined stoichiometry of two molecules of said bi-specific antibody, andone molecule of said targetable construct.

100. The targetable complex of embodiment 97, wherein mixing saidtargetable construct and said bi-specific antibody at relativeconcentrations ranging from about 10⁻³ to about 10³ results in a mixturein which greater than about 99% of the multimeric complexes have adefined stoichiometry of two molecules of said bi-specific antibody, andone molecule of said targetable construct.

101. The targetable complex of embodiment 97, wherein said Kd for saidtarget epitope is from about 0.1 nM to 10 nM.

102. The targetable complex of embodiment 101, wherein said Kd for saidtarget epitope is from about 0.5 nM to about 5 nM.

103. The targetable complex of embodiment 102, wherein said Kd for saidtarget epitope is about 1 nM.

104. The targetable complex of embodiment 97, wherein said molecularscaffold is a peptide or peptide derivative.

105. The targetable complex of embodiment 97, wherein said targetableconstruct is IMP 246.

106. The targetable complex of embodiment 97, wherein said targetableconstruct comprises two constructs that are conjugated to each other,wherein each construct comprises a molecular scaffold and a pair ofcarrier epitopes.

107. The targetable complex of embodiment 107, wherein each of saidconstructs is independently selected from the group consisting of IMP156, IMP 192 and IMP 222.

108. The targetable complex of embodiment 97, wherein said carrierepitope is a hapten.

109. The targetable complex of embodiment 97, wherein said carrierepitope is a chelator, or a complex between a chelator and a metal ion.

110. The targetable complex of embodiment 110, wherein said chelator isselected from the group consisting of DTPA, DOTA, benzyl DTPA, NOTA, andTETA.

111. The targetable complex of embodiment 97, wherein said bi-specificantibody is selected from the group consisting of [hMN]₂-[734scFv]₂ and[hMN^((1253A))]₂-[734scFv]₂.

112. The targetable complex of embodiment 97, wherein at least one armcomprising a binding site for said carrier epitope is 734scFv.

113. The targetable complex of embodiment 97, wherein said targetableconstruct further comprises a bioactive moiety.

114. The targetable complex of embodiment 113, wherein said bioactivemoiety is selected from the group consisting of a drug, a prodrug, anenzyme, a radionuclide, a hormone, an immunomodulator, anoligonucleotide, an image enhancing agent and a toxin.

115. The targetable construct of embodiment 114, wherein said enzyme isselected from the group consisting of malate dehydrogenase,staphylococcal nuclease, delta-V-steroid isomerase, yeast alcoholdehydrogenase, α-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, β-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

116. The targetable construct of embodiment 114, wherein saidimmunomodulator is selected from the group consisting of a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic growth factor, acolony stimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof.

117. The targetable construct of embodiment 114, wherein saidimmunomodulator consists essentially of IL-1, IL-2, IL-3, IL-6, IL-10,IL-12, IL-18, IL-21, G-CSF, GM-CSF, interferon-γ, -α, -β or -γ, TNF-α,and “S1 factor.

118. The targetable construct of embodiment 114, wherein saidoligonucleotide is an anti-sense oligonucleotide.

119. The targetable construct of embodiment 118, wherein said anti-senseoligonucleotide is bcl-2 or p53.

120. A solid support to which the targetable complex of embodiment 97 isbound.

121. A biosensor comprising the targetable complex of embodiment 97.

122. A targetable complex, said targetable complex comprising:

(A) a targetable construct comprising a molecular scaffold and X pairsof carrier epitopes, wherein each of said pairs of carrier epitopes isspecifically bound by one of X bi-specific antibodies, each bi-specificantibody comprising (i) two copies of a first arm comprising a bindingsite for a carrier epitope, and (ii) two copies of a second armcomprising a binding site for one of Y target epitopes, and

-   -   (B) X bi-specific antibodies;    -   wherein:    -   (1) X is a whole integer >3,    -   (2) Y is a whole integer >1,    -   (3) said X bi-specific antibodies can be the same or a mixture        of different bi-specific antibodies,    -   (4) said X pairs of carrier epitopes can be the same or a        mixture of different carrier epitopes, and    -   (5) when Y>2, said Y target epitopes can be the same or a        mixture of different target epitopes;    -   wherein one or more of the following applies:    -   (a) said targetable complex has a Kd of from about 0.1 nM to        about 100 nM for at least one of said target epitopes,    -   (b) a pair of carrier epitopes is simultaneously bound by said        two copies of a first arm comprising a binding site for said        carrier epitope, wherein said two copies of a first arm        comprising a binding site for said carrier epitope are part of        said bi-specific antibody.

123. The targetable construct of embodiment 122, wherein said Kd for atleast one of said target epitopes is from about 0.1 nM to 10 nM.

124. The targetable construct of embodiment 123, wherein said Kd for atleast one of said target epitopes is from about 0.5 nM to about 5 nM.

125. The targetable construct of embodiment 124, wherein said Kd for forat least one of target epitope is about 1 nM.

126. The targetable construct of embodiment 122, wherein said molecularscaffold is a peptide or peptide derivative.

127. The targetable construct of embodiment 122, wherein at least one ofsaid carrier epitopes is a hapten.

128. The targetable construct of embodiment 122, wherein said targetableconstruct further comprises a bioactive moiety.

129. The targetable complex of embodiment 123, wherein said bioactivemoiety is selected from the group consisting of a drug, a prodrug, anenzyme, a hormone, an immunomodulator, an oligonucleotide, aradionuclide, an image enhancing agent and a toxin.

130. The targetable construct of embodiment 129, wherein said enzyme isselected from the group consisting of malate dehydrogenase,staphylococcal nuclease, delta-V-steroid isomerase, yeast alcoholdehydrogenase, α-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, β-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

131. The targetable construct of embodiment 129, wherein saidimmunomodulator is selected from the group consisting of a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic growth factor, acolony stimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof.

132. The targetable construct of embodiment 129, wherein saidimmunomodulator selected from the group consisting of IL-1, IL-2, IL-3,IL-6, IL-10, IL-12, IL-18, IL-21, G-CSF, GM-CSF, interferon-γ, -α, -β or-γ, TNF-α, and “S1 factor.

133. The targetable construct of embodiment 129, wherein saidoligonucleotide is an anti-sense oligonucleotide.

134. The targetable construct of embodiment 133, wherein said anti-senseoligonucleotide is bcl-2 or p53.

135. A solid support to which the targetable construct of embodiment 122is bound.

136. A biosensor comprising the targetable construct of embodiment 122.

137. The targetable construct or the targetable complex of any ofembodiments 1, 31, 57, 72, 97 or 122, wherein said targetable constructor said targetable complex has a half-life in a defined set ofconditions of from about 24 hours to about 500 days.

138. The targetable construct or the targetable complex of embodiment137, wherein said targetable construct or said targetable complex has ahalf-life in a defined set of conditions of from about 24 hours to about10 days.

139. The targetable construct or the targetable complex of embodiment137, wherein said targetable construct or said targetable complex has ahalf-life in a defined set of conditions of from about 24 hours to about72 hours.

140. The targetable construct or the targetable complex of embodiment137, wherein said defined set of conditions is a set of physiologicalconditions.

141. The targetable construct or the targetable complex of embodiment137, wherein said half-life in a defined set of conditions is a serumhalf-life in vitro.

142. The targetable construct or the targetable complex of any ofembodiments 1, 31, 57, 72, 97 or 122, wherein said target epitope isassociated with a hyperproliferative disease.

143. The targetable construct or targetable complex of embodiment 143,wherein said target epitope is a tumor associated antigen associatedwith a type of cancer selected from the group consisting of acutelymphoblastic leukemia, acute myelogenous leukemia, biliary cancer,breast cancer, cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, colorectal cancer, endometrial cancer, esophageal,gastric, head and neck cancer, Hodgkin's lymphoma, lung cancer,medullary thyroid, non-Hodgkin's lymphoma, ovarian cancer, pancreaticcancer, glioma, melanoma, liver cancer, prostate cancer, and urinarybladder cancer.

144. The targetable construct or targetable complex of embodiment 142,wherein said target epitope is a tumor associated antigen selected fromthe group consisting of A3, antigen specific for A33 antibody, BrE3,CD1, CD1a, CD3, CD5, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30,CD45, CD74, CD79a, CD80, HLA-DR, NCA 95, NCA90, HCG and its subunits,CEA, CSAp, EGFR, EGP-1, EGP-2, Ep-CAM, Ba 733, HER2/neu, KC4, KS-1,KS1-4, Le-Y, MAGE, MUC1, MUC2, MUC3, MUC4, PAM-4, PSA, PSMA, RS5, S100,TAG-72, p53, tenascin, IL-6, insulin growth factor-1 (IGF-1), Tnantigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF,17-1 A, an angiogenesis marker, a cytokine, an immunomodulator, anoncogene marker, an oncogene product, and other tumor associatedantigens.

145. A method of treating a hyperproliferative disease, comprisingadministering the targetable construct or targetable complex ofembodiment 142 to a subject in need thereof.

146. The method of embodiment 148, further comprising administering atleast one additional agent suitable for the treatment of saidhyperproliferative disease.

147. The targetable construct or the targetable complex of any ofembodiments embodiments 1, 31, 57, 72, 97 and 122, wherein said targetepitope is associated with a diseased caused by a pathogen.

148. The targetable construct or targetable complex of embodiment 147,wherein said pathogen is selected from the group consisting of abacterium, an intracellular pathogen, a fungus, a parasite and a virus.

149. The targetable construct or targetable complex of embodiment 147,wherein said pathogen is a bacteria selected from the group consistingof Streptococcus agalactiae, Legionella pneumophilia, Streptococcuspyogenes, Escherichia coli, Salmonella typhimurium, Neisseriagonorrhoeae, Neisseria meningitidis, Pneumococcus sp., Hemophilisinfluenzae B, Yersina pestis, Mycobacteria sp., Mycobacterium leprae,Mycobacterium tuberculosis, Treponema pallidum, Pseudomonas aeruginosa,Francisella tularensis, Brucella sp., Brucella abortus, Bacillusanthracis, Clostridium botulinum, and Clostridium tetani.

150. The targetable construct or targetable complex of embodiment 147,wherein said pathogen is an intracellular pathogen selected from thegroup consisting of Chlamydia sp., Rickettsia sp., Leishmania sp.,Kokzidioa sp., Borrelia burgdorfei, Plasmodia sp., Plasmodiumfalciparum, Trypanosoma sp., Trypanosoma brucei and Trypanosoma cruzi.

151. The targetable construct or targetable complex of embodiment 147,wherein said pathogen is a fungus selected from the group consisting ofCandida sp., Candida albicans, Aspergillus sp., Mucor sp., Rhizopus sp.,Fusarium sp., Penicillium marneffei, Microsporum sp., Trichophytonmentagrophytes, Histoplasma capsulatum, Blastomyces dermatitidis andCoccidioides immitis.

152. The targetable construct or targetable complex of embodiment 147,wherein said pathogen is a virus selected from the group consisting ofhepatitis type A, hepatitis type B, hepatitis type C, influenza,varicella, adenovirus, HSV-I, HSV-II, rinderpest, rhinovirous,echovirus, rabies virus, Ebola virus, rotavirus, respiratory syncytialvirus, papilloma virus, papova virus, CMV, echinovirus, arbovirus,huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus,polio virus, HIV-I, HIV-II, Sendai virus, feline leukemia virus,Reovirus, poliovirus, human serum parvo-like virus, SV40, RSV, MMTV,Varicella-Zoster virus, Dengue virus, rubella virus, measles virus,adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murineleukemia virus, VSV, Variola virus, Sindbis virus, lymphocyticchoriomeningitis virus, Rinderpest virus, wart virus and blue tonguevirus.

153. A method of treating a pathogenic disease, comprising administeringthe targetable construct or targetable complex of embodiment 147 to asubject in need thereof

154. A method for ablating non-malignant cells or tissue in a patient,said method comprising treating the patient with the targetableconstruct or targetable complex of any of embodiments 1, 26, 47, 57, 77or 97, wherein said non-malignant cells or tissue are selected from thegroup consisting of ectopic tissue, retained tissue, normal organ tissueand bone marrow.

155. A method of treating a disease in a subject, comprisingadministering to said subject the targetable construct or targetablecomplex of any of embodiments 1, 31, 57, 72, 97 or 122, in an amounteffective to modulate a biochemical process, wherein said target epitopeis comprised within, displayed by or released from one or more cells,tissues, organs or systems of a subject comprising said disease.

156. The method of embodiment 155, wherein said bi-specific antibody isa naked antibody.

157. The method of embodiment 155, wherein modulating said one or morebiochemical processes causes, enhances, limits or prevents cellularquiescence, necrosis, apoptosis or a complement cascade, mutagenesis orcarcinogenesis.

158. The targetable construct or the targetable complex of any ofembodiments embodiments 1, 31, 57, 72, 97 or 122, wherein said targetepitope is associated with a cardiovascular disorder.

159. A method of treating a cardiovascular disorder, comprisingadministering the targetable construct or targetable complex ofembodiment 158 to a subject in need thereof.

160. The method of embodiment 159, further comprising administering atleast one additional agent suitable for the treatment of saidcardiovascular disorder.

161. The targetable construct or the targetable complex of any ofembodiments embodiments 1, 31, 57, 72, 97 or 122, wherein said targetepitope is associated with an autoimmune disorder.

162. A method of treating an autoimmune disorder, comprisingadministering the targetable construct or targetable complex ofembodiment 161 to a subject in need thereof.

163. The method of embodiment 162, wherein autoimmune disorder isselected from the group consisting of acute idiopathic thrombocytopenicpurpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcalnephritis, erythema nodosum, Takayasu's arteritis,Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitisubiterans, Sjogren's syndrome, primary biliary cirrhosis,Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic activehepatitis, polymyositis/dermatomyositis, polychondritis, pamphigusvulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophiclateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia,pemiciousanemia, rapidly progressive glomerulonephritis and fibrosingalveolitis.

164. The method of embodiment 162, further comprising administering atleast one additional agent suitable for the treatment of saidhyperproliferative disease.

165. A pharmaceutical composition comprising the targetable construct ortargetable complex of any of embodiments 1, 31, 57, 72, 97 or 122.

166. A method of treating a disease in a subject, comprisingadministering to said subject the pharmaceutical composition ofembodiment 165.

167. A kit comprising the targetable construct or targetable complex ofany of embodiments 1, 31, 57, 72, 97 or 122.

168. A method of detecting a substance in a sample, comprisingcontacting said sample with the targetable construct or targetablecomplex of any one of embodiments any of embodiments 1, 31, 57, 72, 97or 122, wherein said substance is or comprises a target epitope.

169. A method of detecting a substance in a sample, wherein saidsubstance is or comprises a target epitope, comprising contacting saidsample with the biosensor of any of embodiments 1, 30, 56, 71, 96 or121, and detecting a signal therefrom.

170. The method of embodiment 169, wherein said sample is a biologicalsample.

171. The method of embodiment 170, wherein said biological sample is isselected from the group consisting of a sample of serum, blood, plasma,lymph, urine, feces, skin, intraocular fluid, synovial fluid, phlegm,cartilage and bone, and a biopsy sample.

172. A method of detecting a substance in an environment, wherein saidsubstance is or comprises a target epitope, comprising exposing thebiosensor of any of 1, 30, 56, 71, 96 or 121 to said environment, anddetecting a signal therefrom.

173. A solid support comprising the targetable construct or targetablecomplex of any of embodiments 1, 31, 57, 72, 97 or 122.

174. The solid support of embodiment 173, wherein said solid support isselected from the group consisting of a dipstick, a bead, an interiorsurface of a well in a multiwell plate, and a membrane.

175. A method of purifying a substance, wherein said substance is orcomprises a target epitope, said method comprising contacting a samplecomprising said substance to the solid support of embodiment 173.

176. A method of detecting a substance in a sample, wherein saidsubstance is or comprises a target epitope, said method comprisingcontacting said sample with the solid support of embodiment 173.

177. A dipstick comprising the solid support of embodiment 173.

178. A method of detecting a substance in a fluid sample, wherein saidsubstance is or comprises a target epitope, said method comprisingcontacting said fluid sample with the dipstick of embodiment 177.

179. A multiwell plate comprising the targetable construct or targetablecomplex of any of embodiments 1, 31, 57, 72, 97 or 122.

180. A method of detecting a substance in a sample, wherein saidsubstance is or comprises a target epitope, said method comprisingcontacting said sample with the multiwell plate of embodiment 179.

181. An immunoassay for a substance, wherein said substance is orcomprises a target epitope, said method comprising contacting saidsubstance with the multiwell plate of embodiment 179.

182. A membrane comprising the targetable construct or targetablecomplex of any of embodiments 1, 31, 57, 72, 97 or 122.

183. A method of detecting a substance, wherein said substance is orcomprises a target epitope, said method comprising contacting saidsubstance with the membrane of embodiment 182.

184. A bead comprising the targetable construct or targetable complex ofany of embodiments 1, 31, 57, 72, 97 or 122.

185. A method of purifying a substance, wherein said substance is orcomprises a target epitope, said method comprising contacting saidsubstance with the bead of embodiment 184.

186. A method of binding a substance to a solid support, wherein saidsubstance is or comprises a target epitope, said method comprisingcontacting said substance with the solid support of embodiment 173.

187. A method of removing all or some of a substance from a composition,wherein said substance is or comprises a target epitope, said methodcomprising contacting said composition with the solid support ofembodiment 173.

188. The method of embodiment 187, wherein said composition is a fluid.

189. The method of embodiment 187, wherein said composition is abiological sample.

190. The method of embodiment 189, wherein said sample is a biologicalsample which is selected from the group consisting of serum, blood,plasma, lymph, urine, feces, sweat, intraocular fluid, semen, synovialfluid, mucus, exudent, bone marrow and a biopsy sample.

191. A method of removing all or some of a substance from a tissue in apatient, wherein said substance is or comprises a target epitope, saidmethod comprising contacting said tissue with the solid support ofembodiment 173 and reintroducing said tissue into said patient.

192. The method of embodiment 191, wherein said tissue is selected fromthe group consisting of serum, blood, plasma, lymph, intraocular fluid,bone marrow.

193. The method of embodiment 191, wherein said substance is or is partof a toxin, a pathogen, a hyperproliferative cell and an infected cell.

194. A device for treating a fluid of a patient in order to remove asubstance therefrom and reintroducing said fluid into said patient,wherein said substance is or comprises a target epitope, said devicecomprising the solid support of embodiment 173.

195. A method of treating a patient, said method comprising removing afluid from a patient, passing said fluid through the device ofembodiment 194, and reintroducing said fluid into said patient.

196. The method of embodiment 187, further comprising separating saidsubstance from said solid support.

197. The method of embodiment 187, wherein said substance is anundesirable substance.

198. The method of embodiment 187, wherein said substance is a compoundof interest.

199. The method of embodiment 187, wherein said method is part of amanufacturing process.

200. The method of embodiment 159, wherein said cardiovascular diseaseis an atherosclerotic plaque, ischemia, fibrin clot, vascular clot, andmyocardial infarction.

201. The method of embodiment 200, wherein said vascular clot is anembolus or thrombosis.

202. The method of embodiment 155, wherein said tissue is hypoplastic,absent, anatomically displaced or ectopic.

203. The method of embodiment 155, wherein said disease or disorder is aneurodegenerative or metabolic disease.

204. The method of embodiment 203, wherein said metabolic disease isamyloidosis and said targetable construct binds amyloid.

205. The method of embodiment 203, wherein said neurodegenerativedisease is Alzheimer's disease.

206. A method of diagnosing/detecting a disease in a subject, comprisingadministering to said subject the targetable construct or targetablecomplex of any of embodiments 1, 31, 57, 72, 97 or 122, in an amounteffective to modulate a biochemical process, wherein said target epitopeis comprised within, displayed by or released from one or more cells,tissues, organs or systems of a subject comprising said disease.

207. The method of embodiment 206, wherein said method is suitable fordetecting a cardiovascular lesion.

208. The method of embodiment 207, wherein said method is suitable forphotodynamic diagnosis.

209. The method of embodiment 208, wherein said targetable constructcomprises a photosensitizer selected from the group consisting ofdihematoporphyrin, benzoporphyrin monoacid ring A, tin etiopurpurin,sulfonated aluminum phthalocyanine, and lutetium texaphyrin.

1. A bi-specific antibody comprising the structure [IgG₁]-[scFv]2;wherein said antibody comprises a pair of heavy chains and a pair oflight chains, wherein each heavy chain comprises an IgG1 heavy chain andan scFv, wherein said scFv is fused to the C-terminus of said IgG 1heavy chain, optionally via a linker peptide.
 2. The antibody accordingto claim 1, wherein the binding sites formed by said heavy chain andsaid light chain specifically binds to an epitope on a targeted tissue.3. The antibody according to claim 2, wherein each of said scFv moietiesspecifically binds to a carrier epitope.
 4. The antibody according toclaim 1, wherein said IgG1 is a human, humanized, chimeric, orCDR-grafted antibody.
 5. The antibody according to claim 1, wherein eachof said scFv molecules is human, humanized, or CDR-grafted.
 6. Theantibody according to claim 5, wherein said antibody further comprises abioactive moiety.
 7. The antibody according to claim 6, wherein saidbioactive moiety is selected from the group consisting of a drug, aprodrug, an enzyme, a hormone, an immunomodulator, an oligonucleotide; aradionuclide, an image enhancing agent and a toxin.
 8. The antibodyaccording to claim 1, wherein said antibody is selected from the groupconsisting of [hMN14-IgG1]-[734scFv]₂ and[hMN14-IgG1^((1253A))]-[734scFv]₂.
 9. The antibody according to claim 1,wherein said antibody is selected from the group consisting of[hMN14-IgG1]-[679scFv]₂ and [hMN14-IgG1^((1253A))]-[679scFv]₂.
 10. Theantibody according to claim 1, wherein said antibody is selected fromthe group consisting of [hA20-IgG1]-[734scFv]₂ and[hA20-IgG1^((1253A))]-[734scFv]₂.
 11. The antibody according to claim 1,wherein said antibody is selected from the group consisting of[hA20-IgG1]-[679scFv]₂ and [hA20-IgG1^((1253A))]-[679scFv]₂.
 12. Theantibody according to claim 1, wherein said antibody is selected fromthe group consisting of [hLL2-IgG1]-[734scFv]₂ and[hLL2-IgG1^((1253A))]-[734scFv]₂.
 13. The antibody according to claim 1,wherein said antibody is selected from the group consisting of[hLL2-IgG1]-[679scFv]₂ and [hLL2-IgG1^((1253A))]-[670scFv]₂.
 14. Abinding complex comprising a tetravalent binding molecule bound to atargetable construct, wherein said tetravalent binding moleculecomprises two binding sites for a carrier epitope and two binding sitesfor a target epitope, and wherein said targetable construct comprises amolecular scaffold and at least two carrier epitopes.
 15. The bindingcomplex according to claim 14, wherein said targetable constructcomprises at least two pairs of carrier epitopes and wherein at leasttwo of said tetravalent binding molecules are bound to said targetableconstruct.
 16. The binding complex according to claim 15, wherein saidat least two pairs of carrier epitopes comprise a first pair and asecond pair, wherein said first and second pair are different epitopes,and wherein a first tetravalent binding molecule is bound to said firstpair of carrier epitopes and a second tetravalent binding molecule isbound to said second pair of carrier epitopes.
 17. The binding complexaccording to claim 16, wherein said first and second pair of carrierepitopes are different epitopes.
 18. The binding complex according toclaim 17, wherein said first and second tetravalent binding moleculesbind to the same target epitope.
 19. The binding complex according toclaim 14, wherein said targetable construct is selected from the groupconsisting of IMP 246, IMP 156, IMP 192 and IMP
 222. 20. The bindingcomplex according to claim 14, wherein said carrier epitope is a hapten.21. The binding complex according to claim 14, wherein said carrierepitope is a chelator, wherein said chelator optionally is bound to ametal ion.
 22. The binding complex according to claim 21, wherein saidchelator is selected from the group consisting of DTPA, DOTA, benzylDTPA, NOTA, and TETA.
 23. The binding complex according to claim 14,wherein said tetravalent binding molecule is a bi-specific antibodycomprising the structure [IgG₁]-[scFv]2; wherein said antibody comprisesa pair of heavy chains and a pair of light chains, wherein each heavychain comprises an IgG1 heavy chain and an scFv, wherein said scFv isfused to the C-terminus of said IgG1 heavy chain, optionally via alinker peptide.
 24. The binding complex according to claim 14, whereinsaid molecular scaffold is a peptide or peptide derivative.
 25. Thebinding complex according to claim 14, wherein said target epitope is anantigen associated with a disease.
 26. The binding complex according toclaim 25, wherein said disease is selected from the group consisting ofhyperproliferative disease, pathogenic disease, cancer, cardiovasculardisease, neurodegenerative disease, metabolic disease, and autoimmunedisease
 27. The binding complex according to claim 26, wherein saidtarget epitope is a tumor associated antigen associated with a type ofcancer selected from the group consisting of acute lymphoblasticleukemia, acute myelogenous leukemia, biliary cancer, breast cancer,cervical cancer, chronic lymphocytic leukemia, chronic myelogenousleukemia, colorectal cancer, endometrial cancer, esophageal, gastric,head and neck cancer, Hodgkin's lymphoma, lung cancer, medullarythyroid, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer,glioma, melanoma, liver cancer, prostate cancer, and urinary bladdercancer.
 28. The binding complex according to claim 27, wherein saidtarget epitope is a tumor associated antigen selected from the groupconsisting of A3, antigen specific for A33 antibody, BrE3, CD1, CD1a,CD3, CD5, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD45, CD74,CD79a, CD80, HLA-DR, NCA 95, NCA90, HCG and its subunits, CEA, CSAp,EGFR, EGP-1, EGP-2, Ep-CAM, Ba 733, HER2/neu, KC4, KS-1, KS1-4, Le-Y,MAGE, MUC1, MUC2, MUC3, MUC4, PAM-4, PSA, PSMA, RS5, S100, TAG-72, p53,tenascin, IL-6, insulin growth factor-1 (IGF-1), Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, 17-1A, anangiogenesis marker, a cytokine, an immunomodulator, an oncogene marker,an oncogene product, and other tumor associated antigens.
 29. A methodof treating a disease in a subject, comprising administering to asubject suffering from said disease (i) a tetravalent binding moleculecomprising two binding sites for a carrier epitope and two binding sitesfor a target epitope, wherein said target epitope is an epitopeassociated with said disease, (ii) optionally, a clearing agent, and(iii) a targetable construct comprising a molecular scaffold and atleast two carrier epitopes.
 30. The method according to claim 29,wherein said disease is selected from the group consisting ofhyperproliferative disease, pathogenic disease, cancer, cardiovasculardisease, neurodegenerative disease, metabolic disease, and autoimmunedisease.
 31. The method according to claim 29, wherein said targetableconstruct further comprises a bioactive moiety.
 32. A method ofdiagnosing/detecting a disease in a subject, comprising administering toa subject suspected of suffering from said disease (i) a tetravalentbinding molecule comprising two binding sites for a carrier epitope andtwo binding sites for a target epitope, (ii) optionally, a clearingagent, and (iii) a targetable construct comprising a molecular scaffoldand at least two carrier epitopes, wherein said construct comprises adetectable label.
 33. The method according to claim 32, wherein saidtarget epitope is comprised within, displayed by or released from one ormore cells, tissues, organs or systems of said subject.
 34. A kit,comprising (i) a tetravalent binding molecule comprising two bindingsites for a carrier epitope and two binding sites for a target epitope,(ii) optionally, a clearing agent, and (iii) a targetable constructcomprising a molecular scaffold and at least two carrier epitopes.
 35. Apharmaceutical composition comprising a bispecific antibody according toclaim 1.